Clinical and genetic analysis of a child with Lamb-Shaffer syndrome due to a de novo variant of SOX5 gene

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

To explore the clinical manifestation and genotype of a child with Progressive familial intrahepatic cholestasis 8 (PFIC8) due to variant of KIF12 gene. A child diangosed with PFIC8 at Changzhou Children's Hospital in October 2017 was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples (3 mL each) were collected from the patient, her parents and younger sister. Following extraction of genomic DNA, whole exome sequencing (WES) was carried out. Candidate variants were validated using Sanger sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the pathogenicity of candidate variants was classified. This study has been approved by the Medical Ethics Committee of Changzhou Children's Hospital (Ethics No.: 2023-002). The main clinical manifestations of the child included high GGT cholestasis, portal hypertension, splenomegaly, and abnormal liver enzymes. WES revealed that she has harbored compound heterozygous variants of the KIF12 gene, namely c.245G >A (p.Arg82Gln) and c.1291del (p.Ser431Valfs*13). Bioinformatics analyses showed that both variants were pathogenic. A total of 25 cases were reported in 7 English literature, including 13 males and 12 females. All patients had presented with high GGT cholestasis. Some had progressed to cirrhosis, and 3 cases also had renal lesions. No death was reported. Six children were treated with LTx. Nineteen children were found to harbor homozygous variants of the KIF12 gene, and the remaining six harbored compound heterozygous variants of the same gene. The most common mutation was c.655C>T (p.Arg219*). The mutation sites are mainly located in the Kinesin motor catalytic domain, with high GGT cholestasis as the main clinical feature. No correlation was found between the genotype and phenotype. PFIC8 caused by KIF12 deficiency is mainly characterized by high GGT cholestasis, for which there is no effective treatment. The c.245G>A and c.1291del compound heterozygous variants of the KIF12 gene probably underlay the pathogenesis in this child.

To investigate the clinical characteristics and genetic etiology of a fetus with bilateral ear malformation and microphthalmia. A fetus diagnosed with Syndromic Microphthalmia 5 (MCOPS5) on January 29, 2024 at Ningbo Women and Children's Hospital was selected as the study subject. A retrospective study was conducted to collect clinical data. Peripheral blood samples (3 mL) were collected from the parents, and amniotic fluid (10 mL) was obtained from the fetus. Genomic DNA was extracted and subjected to whole-exome sequencing (WES). Candidate variants were validated by Sanger sequencing of the family members. The pathogenicity of the candidate variant was classified according to the guidelines from the American College of Medical Genetics and Genomics (ACMG). This study was approved by the Ethics Committee of Ningbo Women and Children's Hospital (Ethics No.: EC2023-094). The gestational age of the fetus was 23+2 weeks. Prenatal magnetic resonance imaging (MRI) revealed hypoplastic left external ear, bilateral reduced eyeball volume, and abnormal brain parenchyma development. WES has identified a heterozygous frameshift variant in the OTX2 gene (NM_021728.4: c.706_725del, p.Thr236ProfsTer17). Sanger sequencing confirmed that neither parent has carried the same variant, indicating a de novo origin. According to the ACMG guidelines, this variant was classified as likely pathogenic (PVS1_Strong+PM2_Supporting+PS2_Supporting). The heterozygous frameshift variant (NM_021728.4: c.706_725del) of the OTX2 gene probably underlay the pathogenesis of this fetus. Above finding has expanded the mutational spectrum of OTX2 gene and may contribute to the understanding of syndromic microphthalmia.

To explore the clinical characteristics and genetic etiology of a child with Osteopathia striata with cranial sclerosis (OSCS) due to variant of AMER1 gene. A child presented at the Affiliated Children's Hospital of Zhengzhou University in July 2024 due to growth and development retardation was selected as the study subject. A retrospective study was conducted to collect the child's clinical data. Peripheral blood samples (2 mL each) were collected from the child and her parents, and genomic DNA was extracted for whole exome sequencing (WES). Sanger sequencing was used for the verification of candidate variants. The pathogenicity of variant was rated according to the guidelines from American College of Medical Genetics and Genomics (ACMG). The study has been approved by the Medical Ethics Committee of the Children's Hospital Affiliated to Zhengzhou University (Ethics No.: 2024-108-001). The patient, a 4-year-and-10-month-old girl, presented with global developmental delay, short stature, cleft palate, distinct facial features, and hearing impairment. WES revealed that she has harbored a heterozygous c.790_794dup (p.Cys265Trpfs*19) variant of the AMER1 gene, which was not detected in either parent. Based on the guidelines from ACMG, the gene variant was classified as pathogenic (PVS1 + PS2 + PM2_supporting). As the result of a non-triplet base insertion in the coding region of the AMER1 gene, it has converted a codon originally encoding an amino acid into a stop codon, and led to a truncated protein, causing severe alteration and dysfunction of the protein. The child was diagnosed with OSCS for clinical features such as global developmental delay, short stature, cleft palate, distinctive facial features, and hearing impairment, for which the de novo heterozygous frameshift variant AMER1: c.790_794dup (p.Cys265Trpfs*19) may be accountable. Above finding has expanded the mutational spectrum of OSCS and provided a basis for genetic counseling and prenatal diagnosis for the family.

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

To explore the clinical and genetic characteristics of a boy with X-linked familial Behcet-like autoinflammatory syndrome-2 (AIFBL2). A boy who was admitted to Children's Hospital Affiliated to Zhengzhou University in December 2023 due to recurrent oral ulcers for 2 years, intermittent abdominal pain and fever for more than 1 year was selected as the study subject. Clinical data of the patient was collected. Whole exome sequencing (WES) was carried out, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. A literature search was conducted in OMIM, PubMed, Wanfang Data Knowledge Service Platform, China Biomedical Literature Service System, and the VIP database using the keywords "ELF4 gene" "deficiency in ELF4, X-linked" "ELF4 deficiency" and "DEX" to identify recently published studies. This study was approved by the Medical Ethics Committee of the Children's Hospital Affiliated to Zhengzhou University (Ethics No.: 2023-H-K44). The patient, a 12-year-old male, presented with recurrent mouth ulcers, fever and abdominal pain. Lymphocyte subsets showed a significant decrease in NK cells. Abdominal CT showed thickening of local intestinal wall in the lower right abdomen. Colonoscopy revealed a solitary deep longitudinal ulcer in the ileocecal region. Genetic testing revealed a hemizygote missense variant c.687C>G, with his mother showing the same mutation at this locus. According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), the variant was considered likely pathogenic (PP1+PP2+PM2_Supporting+PP3+PP4). Literature review has found 19 AIFBL2 patients including 1 patient from this study. Mouth ulcer, fever, rash and abdominal pain were the primary clinical manifestations, for which genetic testing is the main diagnostic method. The hemizygote c.687C>G missense variant of the ELF4 gene probably underlay the AIFBL2 in this child, which has provided a basis for his clinical diagnosis and genetic counseling.

DNA-based screening and population health: a points to consider statement for programs and sponsoring organizations from the American College of Medical Genetics and Genomics (ACMG)

To explore the clinical, genetic, therapeutic and prognostic characteristics of two children with Lesch-Nyhan syndrome (LNS) in order to enhance understanding of this disease and formulate more effective therapeutic strategies. Clinical data were collected from two children clinically diagnosed with LNS who were treated at Anhui Provincial Children's Hospital from April 2023 to January 2024. Data were retrospectively collected and included clinical manifestations (symptoms, signs, laboratory and imaging findings), treatment course, and results of follow-up. Peripheral venous blood samples were obtained from child 1 and his parents. Whole-exome sequencing (WES) was performed. Candidate variants were validated by Sanger sequencing. Standard bioinformatic analysis of the raw WES data was conducted, including quality control, alignment, variant calling, and annotation. Candidate pathogenic variants were filtered using population frequency databases (e.g., gnomAD), disease databases (e.g., OMIM, ClinVar), and multiple in silico pathogenicity prediction tools (e.g., SIFT, PolyPhen-2, CADD). Phenotype matching was integrated using Human Phenotype Ontology (HPO) terms. Pathogenicity classification of variants was performed according to the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines for the Interpretation of Sequence Variants (2015). This study was approved by the Medical Ethics Committee of Anhui Children's Hospital, Children's Hospital of Fudan University (Ethics No.: EYLL-2014-027). Child 1, a 4-year-old boy, had presented with developmental delay for over 3 years, accompanied by abnormal postures and involuntary lip-biting. Physical examination revealed limb dystonia, anxious expression, lower lip damage, and communication difficulties. Laboratory tests showed hyperuricemia and renal stones. Genetic testing identified a hemizygote variant of the HPRT1 gene, c.135G>T (p.Arg45Ser), inherited from an asymptomatic carrier mother, which confirmed the diagnosis of LNS. This variant was absent from population databases (gnomAD, 1000 Genomes, dbSNP). Protein function prediction tools consistently indicated it as a pathogenic or likely pathogenic variant (SIFT, PolyPhen-2, CADD, and REVEL scores all reached pathogenic thresholds). Protein structural modeling revealed that the variant may disrupt the hydrogen-bonding network compromising the tetramer stability. ACMG classification designated it as likely pathogenic (PM1+PM2_Supporting+PM5+PP3). The patient was treated with benhaxol hydrochloride, baclofen, and clonazepam to improve his neurological symptoms, in addition with treatment with febuxostat from the Nephrology Department to manage his purine metabolism. After one year of follow-up, the patient's abnormal posture showed slight improvement, self-injurious behavior persisted but was managed with protective gloves, blood uric acid levels normalized, and renal stones decreased. Case 2, a 13-year-old boy, was hospitalized to the Nephrology Department due to urinary tract infection. Following successful control of the infection, his limb dystonia has worsened, leading to his transfer to the Neurology Ward. The patient had a history of delayed motor and language development, abnormal postures, and lip-biting self-injurious behavior, with elevated blood uric acid levels, leading to the diagnosis of LNS. His parents had declined genetic testing due to financial constraints. Following discharge, the patient did not adhere to the prescribed medication regimen or attend scheduled outpatient visits. The patient had died by the time of the 4-month follow-up contact. Variants of the HPRT1 gene probably underlay the LNS in the two children, and the HPRT1 is the only known pathogenic gene for LNS. Early genetic diagnosis, strict adherence to multidisciplinary comprehensive treatment, and intensive intervention for self-injurious behaviors are crucial for improving the quality of life and prolonging the survival of children with LNS.

To explore the genotype-phenotype correlation in a Charcot-Marie-Tooth type 2A2A (CMT2A2A) pedigree and to provide genetic counseling for its subsequent pregnancies. A Chinese pedigree presenting with "lower limb muscle atrophy and movement disorders" at the Prenatal Diagnosis Center of Xuzhou Central Hospital between January and August 2024 was selected as the study subject. Relevant clinical data were collected from the pedigree members. Peripheral blood samples from affected individuals, and amniotic fluid and/or chorionic villus samples were obtained for DNA extraction. Whole exome sequencing (WES) was carried out. Candidate variants were verified by Sanger sequencing. Pathogenicity assessment and bioinformatic analysis were conducted. This study was approved by the Medical Ethics Committee of Xuzhou Central Hospital (Ethics No. XZXY-LK-20240111-0019). All affected individuals in this pedigree were females, whom included the proband, her mother, and her first daughter. Earlier age of onset was associated with more severe lower limb atrophy. A heterozygous missense variant of the MFN2 gene, namely c.314C>T (p.Thr105Met), was identified in the proband, her mother, daughter, and the third fetus from a re-marriage. The same variant was absent in her elder brother, current husband, and her fourth fetus. Based on the guidelines from American College of Medical Genetics and Genomics (ACMG) and recommendations from Clinical Genome Resources (ClinGen), the variant was classified as pathogenic (PP1_Strong+PM1+PS3+PS4_Moderate+PP3_Moderate+PM2_Supporting). Analyses with PROVEAN and Mutation Taster had categorized the variant as "deleterious" and "disease-causing", respectively. Analysis with Uniprot and Jalview showed that the affected amino acid residue is conserved across multiple species. ChEBI software predicted that the variant may alter the polarity of the 105th amino acid residue. The c.314C>T (p.Thr105Met) missense variant of the MFN2 gene probably underlie the CMT2A2A in this pedigree. Above finding has enabled prenatal diagnosis and genetic counseling for its subsequent pregnancies.

To investigate the clinical and genetic characteristics of a Chinese pedigree affected with Neurodevelopmental disorder with absent language and variable seizures (NEDALVS) due to variant of WASF1 gene, and to review the literature on NEDALVS associated with WASF1 gene variants. A 4-year-and-8-month-old boy with NEDALVS diagnosed at Linyi People's Hospital in July 2024 due to "discovering language development delay for more than 2 years" and his family members were selected as the study subjects. Clinical data of the family members were collected. Peripheral venous blood samples were collected from family members. Whole-exome sequencing (WES) was performed, and candidate variants were verified, by Sanger sequencing. Pathogenicity of candidate variant was classified according to the Standards and Guidelines for the Interpretation of Sequence Variants established by the American College of Medical Genetics and Genomics (ACMG). Using the MUpro website, SWISS-MODEL, PyMOL, Clustal X, PolyPhen-2, and Mutation Taster software, bioinformatics analysis of protein three-dimensional structure modeling for gene mutations, cross-species conservation of mutant amino acids, and pathogenicity prediction of mutation sites. Relevant literature was retrieved from databases such as CNKI, Wanfang Data Knowledge Service Platform, and PubMed, and the clinical phenotypes and genotypes of patients with WASF1 gene mutations reported in the literature were summarized and analyzed. This study was approved by the Medical Ethics Committee of Linyi People's Hospital (Ethics No.: YX200303). The proband, a 4-year and 8-month-old male, mainly presented with delayed language and motor development, accompanied by autistic behaviors; the proband's younger brother was 2 years and 7 months old at the time of consultation, mainly presented with delayed language and motor development, accompanied by short stature; the proband's mother mainly presents with limited language expression and poor interpersonal interaction; the proband's maternal grandmother mainly presents with soliloquizing?behavior. The results of WES showed that the proband carried a heterozygous mutation c.214C>T (p.Arg72Cys) in the WASF1 gene, and this site has not been recorded in the database. Sanger sequencing confirmed that the proband's younger brother, mother, and maternal grandmother had harbored the same variant. Based on the guidelines from the ACMG, this variant was rated as likely pathogenic (PM2_Supporting+PP1+PP3+PP4). Through SWISS-MODEL homology modeling and PyMOL structure visualization analysis, it was further confirmed that this variant can lead to a decrease in protein stability. Amino acid sequence conservation analysis of the WASF1 protein using Clustal X software suggested that the c.214C>T (p.Arg72Cys) variant has caused replacement of a highly conserved amino acid. According to the results of PolyPhen-2 and Mutation Taster, the p.Arg72Cys variant was predicted to be a hazardous. By following the retrieval strategy set in this study, a total of 5 research articles regarding to patients with NEDALVS caused by WASF1 gene mutations were retrieved, which involved 15 patients. Combining the proband and their family members discovered in this study, there were a total of 19 NEDALVS patients. The main clinical features included: motor developmental delay (100%, 17/17), language/intellectual developmental delay (100%, 17/17), epilepsy (64.7%, 11/17), autistic behavior (76.5%, 13/17), hypotonia (70.6%, 12/17), abnormal electroencephalogram (64.7%, 11/17), and short stature (17.6%, 3/17). All 19 patients had heterozygous mutations, with 8 mutation sites. Missense mutations were the most common, accounting for 84.2% (16/19). A pathogenic variant of the WASF1 gene was identified in a pedigree affected with NEDALVS. Discovery of the novel variant has, expanded the mutational spectrum of the WASF1 gene.

To explore the genetic etiology of a child with Brain small vessel disease 1 with ocular anomalies. A child who was admitted to Ningbo Women and Children's Hospital on May 28, 2022 was selected for the study. Clinical data were collected, and peripheral blood samples from the child and her parents were obtained for genomic DNA extraction. Whole exome sequencing (WES) was performed to screen for pathogenic variants. Candidate variants were validated via Sanger sequencing and subjected to bioinformatic analysis. This study was approved by the Medical Ethics Committee of Ningbo Women and Children's Hospital (Ethics No. EC2020-014). The child was a 7-year-old female with a diagnosis of epilepsy. WES revealed that she has carried a heterozygous missense variant in the COL4A1 gene: c.1792G>A (p.Gly598Ser). Sanger sequencing confirmed that her parents both had the wild-type genotype for this variant. Based on American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines for the Interpretation of Sequence Variants, the variant were predicted to be a likely pathogenic (PS2+PM1+PM2_Supporting+PP3). Bioinformatics predicted that amino acid 598 was highly conserved in different species, formed hydrogen bond with Asp599 after becoming Ser598. The heterozygous missense variant of the COL4A1 gene c.1792T>C (p.G598S) could be the pathogenic cause of this child with Brain small vessel disease 1 with ocular anomalies.

To explore the clinical phenotype and variants of KIF4A gene associated with X-linked intellectual disability type 100 (XLID100) in a child by whole-exome sequencing (WES). A child presented at the Children's Hospital Affiliated to Nanjing Medical University in September 2023 was selected as the study subject. Clinical data of the child was retrospectively analyzed. Peripheral blood samples were collected from the child and his family members for WES analysis. Candidate variant was verified by Sanger sequencing. Pathogenicity of the candidate variant was rated based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). The variant was also searched in dbSNP, OMIM, HGMD, ClinVar and gnomAD databases. Amino acid sequences of the KIF4A protein across various species were retrieved from the Ensembl Genome Browser Database and analyzed using Clustal Omega software. Relevant literature on KIF4A gene mutations associated with XLID100 was reviewed. This study has been approved by the Medical Ethics Committee of the Hospital (Ethics No. 202402022-1). The child, a 3-year-6-month-old male, had manifested intellectual impairment, language delay, autism, and choroid cyst revealed by cranial magnetic resonance imaging. No facial dysmorphism, tooth anomaly, gross motor development delay or regression, and history of seizure and febrile convulsion was noted. WES revealed that he has harbored a c.3385delinsTATC (p.Thr1129delinsTyrPro) variant of the KIF4A gene. Sanger sequencing confirmed that his mother and sister have harbored the same variant, whilst his father was of the wild type. Both of his parents had a normal phenotype. The variant was classified as of uncertain significance based on the guidelines from the ACMG. It was not recorded by the dbSNP, OMIM, HGMD, ClinVar and the gnomAD database. Conservative analysis suggested that the variant site, which normally encodes a cysteine, is highly conserved among various species. A review of the literature had retrieved 6 relevant articles documenting a total of 27 cases of KIF4A gene mutations, with only one case from China. The c.3385delinsTATC (p.Thr1129delinsTyrPro) variant of the KIF4A gene probably underlay the XLID100 in this child. Above finding has provided a reference for the clinical diagnosis and genetic counseling and enriched the mutation spectrum of the KIF4A gene.

To explore the clinical phenotype and genetic characteristics of two siblings with intellectual disability. Clinical data and peripheral blood samples were collected from the proband, his younger sister and parents whom had presented at Wenzhou Central Hospital in February 2024. Low-coverage massively parallel copy number variation sequencing (CNV-seq) and whole exome sequencing (WES) were carried out for the family. Candidate variants were verified by Sanger sequencing. Prenatal diagnosis was performed on a fetus upon the couple's subsequent pregnancy. The study was approved by the Medical Ethics Committee of Wenzhou Central Hospital (Ethic No. L2024-07-001). The proband was a 12-year-old boy who had presented with mental retardation and language delay. His 10-year-old sister also manifested delayed mental and motor development. Whole exome sequencing revealed that the proband and his sister had respectively harbored a novel heterozygous c.3549_3550del (p.Glu1183Aspfs*29) variant of the TRIP12 gene and a novel heterozygous c.99del (p.Ser34Alafs*38) variant of the GRIN2B gene. Sanger sequencing confirmed that both variants had a de novo origin. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were classified as pathogenic (PVS1+PS2_Supporting+PM2_Supporting). Neither variant was found to be carried by the fetus upon prenatal diagnosis. Above variants probably underlay the mental disorders in the two siblings, and the concurrent occurrence of two novel pathogenic variants in a family has been extremely rare.

To explore the genetic etiology for a child presenting with motor retardation, language delay, intellectual disability, and dysmorphic features. A child presented at Linyi People's Hospital in June 2022 was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples were obtained from the child and her parents. Following extraction of genomic DNA, whole-exome sequencing (WES) was carried out. Candidate variant was validated by Sanger sequencing. Amniotic fluid samples were obtained from the mother's subsequent pregnancies for prenatal diagnosis. This study has been reviewed and approved by the Medical Ethics Committee of Linyi People's Hospital (Ethics No.: 2019-134). The proband was a 2-year-old girl showing developmental delays in motor, language, and intellectual domains, strabismus, hypertelorism, hearing impairment, obesity, and brachymesophalangy of the fifth finger. Magnetic resonance imaging revealed abnormalities of the white matter. Chromosomal microarray analysis (CMA) identified a 15q26.3 duplication (chr15:101562020_102060896 × 3) inherited from her mother. WES has uncovered a heterozygous c.1931A>G (p.Tyr644Cys) variant in the FBXO11 gene. Sanger sequencing confirmed the variant to be de novo in origin. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic. Prenatal diagnosis revealed that the fetuses from the mother's second and third pregnancies did not harbor the same variant. The c.1931A>G (p.Tyr644Cys) variant of the FBXO11 gene probably underlay the abnormal phenotype in the child. Based on its genotype and phenotype, the proband was diagnosed with Intellectual developmental disorder with dysmorphic facies and behavioral abnormalities.

To explore the genetic etiology of a child with Spastic paraplegia and psychomotor retardation with or without seizures (SPPRS). A child who was admitted to the Children's Hospital Affiliated to Nanjing Medical University in April 2022 for motor developmental delay, intellectual disability, and hypertonia was selected as the study subject. Relevant clinical data were retrospectively analyzed. Whole exome sequencing (WES) was carried out for the child and his parents. Candidate variants were searched in the Single Nucleotide Polymorphism Database (dbSNP) and Online Mendelian Inheritance in Man (OMIM) database. Pathogenicity of the variants was assessed based on guidelines from the American College of Medical Genetics and Genomics (ACMG). Using key words such as "HACE1 gene" "Spastic paraplegia and psychomotor retardation with or without seizures" and "SPPRS", previous reports on SPPRS patients due to HACE1 gene variants were retrieved from the CNKI, Wanfang Data Knowledge Service Platform, CQVIP, and PubMed databases, with the time set from January 1, 2000 to April 7, 2024. A mutation map for the HACE1 protein in the patients was created. This study was approved by the Ethics Committee of the Children's Hospital Affiliated to Nanjing Medical University (Ethics No. 202404008-1). The clinical manifestations of the child had included motor developmental delay, intellectual disability and hypertonia. Magnetic resonance imaging revealed hypoplasia of posterior corpus callosum and splenium, with slight enlargement of lateral ventricles. WES revealed that the child has harbored compound heterozygous variants of the HACE1 gene, namely c.535(exon7)_c.538(exon7)delACAG (p.T179fs*5) and c.1678+2(IVS15)T>C, which were respectively inherited from his parents. Based on the guidelines from the ACMG, the variants were respectively rated as likely pathogenic (PVS1 + PM2_Supporting) and pathogenic (PVS1 + PM2_Supporting + PM3). Literature search has identified 8 papers, which reported 23 SPPRS cases due to HACE1 gene variants. All patients exhibited psychomotor developmental delay, among whom 18 HACE1 gene variants were identified. The c.535(exon7)_c.538(exon7)delACAG (p.T179fs*5) and c.1678+2(IVS15)T>C compound heterozygous variants of the HACE1 gene probably underlay the pathogenesis of SPPRS in this child. Above discovery has enriched the mutational and phenotypic spectrum of the HACE1 gene and provided a reference for clinical diagnosis and genetic counseling.