Genotype-phenotype Correlation Research Articles

ATP1A3 Disease Spectrum Includes Paroxysmal Weakness and Encephalopathy Not Triggered by Fever.

Heterozygous pathogenic variants in ATP1A3, which encodes the catalytic alpha subunit of neuronal Na+/K+-ATPase, cause primarily neurologic disorders with widely variable features that can include episodic movement deficits. One distinctive presentation of ATP1A3-related disease is recurrent fever-triggered encephalopathy. This can occur with generalized weakness and/or ataxia and is described in the literature as relapsing encephalopathy with cerebellar ataxia. This syndrome displays genotype-phenotype correlation with variants at p.R756 causing temperature sensitivity of ATP1A3. We report clinical and in vitro functional evidence for a similar phenotype not triggered by fever but associated with protein loss-of-function. We describe the phenotype of an individual with de novo occurrence of a novel heterozygous ATP1A3 variant, NM_152296.5:c.388_390delGTG; p.(V130del). We confirmed the pathogenicity of p.V130del by cell survival complementation assay in HEK293 cells and then characterized its functional impact on enzymatic ion transport and extracellular sodium binding by two-electrode voltage clamp electrophysiology in Xenopus oocytes. To determine whether variant enzymes reach the cell surface, we surface-biotinylated oocytes expressing N-tagged ATP1A3. The proband is a 7-year-old boy who has had 2 lifetime episodes of paroxysmal weakness, encephalopathy, and ataxia not triggered by fever. He had speech regression and intermittent hand tremors after the second episode but otherwise spontaneously recovered after episodes and is at present developmentally appropriate. The p.V130del variant was identified on clinical trio exome sequencing, which did not reveal any other variants possibly associated with the phenotype. p.V130del eliminated ATP1A3 function in cell survival complementation assay. In Xenopus oocytes, p.V130del variant Na+/K+-ATPases showed complete loss of ion transport activity and marked abnormalities of extracellular Na+ binding at room temperature. Despite this clear loss-of-function effect, surface biotinylation under the same conditions revealed that p.V130del variant enzymes were still present at the oocyte's cell membrane. This individual's phenotype expands the clinical spectrum of ATP1A3-related recurrent encephalopathy to include presentations without fever-triggered events. The total loss of ion transport function with p.V130del, despite enzyme presence at the cell membrane, indicates that haploinsufficiency can cause relatively mild phenotypes in ATP1A3-related disease.

Choroidal neovascularisation secondary toX-linked retinoschisis

AimsChoroidal neovascularisation (CNV) in patients with X-linked retinoschisis (XLRS) has been poorly documented. This study aims to investigate the prevalence and clinical characteristics of CNV in patients with XLRS, as...

30years of prophylactic thyroidectomy for hereditary medullary thyroid cancer : Amilestone in translational medicine

Medullary thyroid cancer (MTC) is the most frequent manifestation of multiple endocrine neoplasia type 2 (MEN2) that determines the oncological outcome. Germline mutations in the rearranged during transfection (RET) protooncogene, atumor suppressor gene on chromosome 10q11.2, were identified 30 years ago as the genetic basis of MEN2 and published in 1993 and 1994. These seminal findings gave rise to the concept of prophylactic thyroidectomy for asymptomatic gene mutation carriers based on apositive RET gene test, which has become the standard of care ever since. Clinical genetic investigations showed genotype-phenotype correlations with respect to the individual gene mutation regarding the penetrance and onset of MTC and to alesser extent also with respect to the other components of MEN2, pheochromocytoma and primary hyperparathyroidism. From this a clinically relevant risk stratification could be derived. Initially, the optimal timing of prophylactic thyroidectomy was primarily based on the RET genotype alone, which was not sufficient for a precise age recommendation and subsequently required additional consideration of calcitonin serum levels for fine tuning. Calcitonin levels first show the risk of lymph node metastasis when they exceed the upper normal limit of the assay independent of carrier age and RET mutation. Routine calcitonin screening of patients with nodular thyroid disease, screening of families on identification of MEN2 index patients, and pre-emptive thyroidectomy in carriers of gene mutations with normal calcitonin levels have led to the fact that nowadays, 30years after the first description of the gene mutations causing the disease, the life-threatening hereditary MTC has become curable: ashining example for the success of translational transnational medical research for the benefit of patients.

Whole-Genome Sequencing Identified a Novel Mutation in the N-Terminal Domain of KIF5A in Chinese Patients with Familial Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by progressive damage to both upper and lower motor neurons. Genetic factors are known to play a crucial role in ALS, as genetic studies not only advance our comprehension of disease mechanisms but also help unravel the complex phenotypes exhibited by patients. To gain further insights into the genetic landscape of ALS in the Chinese population and explore genotype–phenotype correlations among individuals, we conducted whole-genome sequencing to screen genes in 34 Chinese familial ALS (FALS) probands lacking the most common ALS-associated genes. Within this cohort, we identified a rare heterozygous missense mutation in the N-terminal domain of KIF5A (c.86A>G) in one of the probands. This finding is significant as mutations in the KIF5A gene have been implicated in ALS in European cohorts since 2018, predominantly characterized by C-terminal mutations. Analysis of the clinical phenotype within this familial lineage revealed a delayed onset of symptoms, an extended survival duration, and initial manifestations in both upper limbs. These observations underscore the clinical heterogeneity observed in ALS patients harboring KIF5A mutations. In conclusion, our study contributes to the growing body of evidence linking KIF5A to ALS and enhances our understanding of the intricate genetic landscape of this disease.

De novo variation in ARID1B gene causes Coffin-Siris syndrome 1 in a Chinese family with excessive early-onset high myopia

Coffin-Siris syndrome (CSS) is a rare autosomal dominant inheritance disorder characterized by distinctive facial features, hypoplasia of the distal phalanx or nail of the fifth and additional digits, developmental or cognitive delay of varying degree, hypotonia, hirsutism/hypertrichosis, sparse scalp hair and varying kind of congenital anomalies. CSS can easily be misdiagnosed as other syndromes or disorders with a similar clinical picture because of their genetic and phenotypic heterogeneity. We describde the genotype-phenotype correlation of one patient from a healthy Chinese family with a novel genotype underlying CSS, who was first diagnosed in the ophthalmology department as early-onset high myopia (eoHM). Comprehensive ophthalmic tests as well as other systemic examinations were performed on participants to confirm the phenotype. The genotype was identified using whole exome sequencing, and further verified the results among other family members by Sanger sequencing. Real-time quantitative PCR (RT-qPCR) technology was used to detect the relative mRNA expression levels of candidate genes between proband and normal family members. The pathogenicity of the identified variant was determined by The American College of Medical Genetics and Genomics (ACMG) guidelines. STRING protein-protein interactions (PPIs) network analysis was used to detect the interaction of candidate gene-related proteins with high myopia gene-related proteins. The patient had excessive eoHM, cone-rod dystrophy, coarse face, excessive hair growth on the face, sparse scalp hair, developmental delay, intellectual disability, moderate hearing loss, dental hypoplasia, patent foramen ovale, chronic non-atrophic gastritis, bilateral renal cysts, cisterna magna, and emotional outbursts with aggression. The genetic assessment revealed that the patient carries a de novo heterozygous frameshift insertion variant in the ARID1B c.3981dup (p.Glu1328ArgfsTer5), which are strongly associated with the typical clinical features of CSS patients. The test results of RT-qPCR showed that mRNA expression of the ARID1B gene in the proband was approximately 30% lower than that of the normal control in the family, suggesting that the variant had an impact on the gene function at the level of mRNA expression. The variant was pathogenic as assessed by ACMG guidelines. Analysis of protein interactions in the STRING online database revealed that the ARID1A protein interacts with the high myopia gene-related proteins FGFR3, ASXL1, ERBB3, and SOX4, whereas the ARID1A protein antagonizes the ARID1B protein. Therefore, in this paper, we are the first to report a de novo heterozygous frameshift insertion variant in the ARID1B gene causing CSS with excessive eoHM. Our study extends the genotypic and phenotypic spectrums for ARID1B-CSS and supplies evidence of significant association of eoHM with variant in ARID1B gene. As CSS has high genetic and phenotypic heterogeneity, our findings highlight the importance of molecular genetic testing and an interdisciplinary clinical diagnostic workup to avoid misdiagnosis as some disorders with similar manifestations of CSS.

#1792 Phenotypic variability in PAX2-related nephropathies: a monocentric study

Abstract Background and Aims Pax2 mutations are known to cause kidney disease; both syndromic conditions, as kidney coloboma syndrome, and kidney specific disorders, as FSGS, have been described. Due to the rarity of Pax2-related nephropathies, data on genotype-phenotype correlation are limited. Method This observational and mono-centric study describes three families (A, G, P) referring to our Nephrology center, carrying Pax2 mutations. A total of 12 subjects were investigated: 2 individuals in family A, 4 in family G and 6 in the family P. Genetic analysis was carried out by NGS; detected mutations were confirmed by Sanger sequencing. Results Family A It includes 2 affected subjects, a 40 year-old woman and her mother (Fig. 1A). The mother had chorioretinic coloboma and was under hemodialytic therapy since the age of 50. The daughter had chorioretinic coloboma and bilateral renal hypodysplasia, proteinuria and a progressive decline of the eGFR since 34 years; renal biopsy excluded the presence of GN (Table 1). Both have the heterozygote c.890delC variant (p.Thr297fs) in Pax2. Family G It includes 4 affected individuals belonging to 3 different generations (Fig. 1B). The disorder was discovered after the evidence of advanced renal failure in a pregnant 27 y-old lady. The study of her family revealed the presence of kidney disease in her sister (transplanted on young age) and her father; one of her children has the mutation (Table 1). The PAX2 c.194T>C(p.Val65Ala HET) mutation segregates with the phenotype in the family. Family P It includes 6 affected individuals of 3 different generations (Fig. 1C); the index-case presented with heavy proteinuria: kidney biopsy revealed a C3 nephropathy. His son was transplanted when 18 years old for bilateral renal hypoplasia. His mother died after some years of dialysis (Table 1). PAX2 mutation on exon3: c.361G>A(p.Glu121Lys HET). Conclusion This case series demonstrates the phenotypic variability of patients with Pax2 mutations and suggests the utility of new generation genetics (NGS) to get the diagnosis in atypical conditions. The implementation of international registers and basic studies will be indispensable to define the mechanisms involved in the development of the disease and to address the reasons of clinical variability.

#844 Renal manifestations of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome: an updated systematic review

Abstract Background and Aims Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a systemic autoimmune genetic disorder caused by mutation of the forkhead box protein 3 (FOXP3) gene. We performed this study to analyze the clinical and demographic characteristics of renal involvement in IPEX patients, genotype-phenotype correlations and the effect of renal involvement on patient outcome. Method We performed a literature search (PubMed and EMBASE) to systematically investigate the case reports of IPEX with renal involvement which were published before Dec 21st, 2023. Results A total of 37 patients were identified. All IPEX patients included had FOXP3 mutations which were most frequently located in the forkhead domain. Among 25 patients in whom detailed information is available, 22 showed single presentation (6 proteinuria, 8 nephrotic syndrome (nephrotic range proteinuria), 3 acute glomerulonephritis, 5 renal insufficiency), 2 showed multiple presentations (1 nephrotic range proteinuria, microscopic hematuria and 1 nephrotic range proteinuria, microscopic hematuria, renal insufficiency). Renal biopsy was documented in 19 patients. Membranous nephropathy (n = 9) was most common, followed by Interstitial nephritis (n = 3), membranoproliferative nephropathy (n = 2), tubulopathy (n = 2), mesangial proliferative glomerulonephritis (n = 2), autoimmune nephritis (n = 1). Nephrotic syndrome was more frequent in patients with intron 6 mutation (p < 0.05). However, renal presentation of IPEX syndrome was not related to patient outcome (death). Conclusion We found renal involvement is one of the important manifestations of IPEX syndrome and membranous nephropathy was most common. Close renal monitoring is necessary to detect renal involvement of IPEX syndrome. Further studies are necessary to compare the course of glomerulonephritis between IPEX-related and IPEX-unrelated one. Response to treatment should also be elucidated in patients with IPEX in the future.

#662 Characterization of novel gene mutation sites associated with autosomal dominant polycystic kidney disease on 19 chromosomes

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the most common single-gene inherited kidney disorder, affecting approximately 1 in 500-1000 newborns and often leading to end-stage renal disease. It involves cyst formation in various organs, with renal manifestations including cysts, hematuria, kidney stones, proteinuria, and hypertension. Liver, pancreas, seminal vesicles, spleen, and intracranial aneurysms may also be affected. Genetic diagnosis, particularly for patients without a family history or with atypical imaging, is considered the gold standard. ADPKD is associated with several pathogenic genes, such as PKD1 and PKD2, as well as others like HNF-1B, GANAB, DNAJB11, SEC63, ALG8, ALG9, PRKCSH, LRP5, SEC61B, and SEC63. Understanding the genetic basis is crucial for early diagnosis and identifying high-risk individuals. This study, based on genetic data from patients with renal polycystic kidney disease at Zhong Da Hospital Southeast University, identified novel ADPKD gene mutations. It explored structural changes in proteins, predicting effects on phenotype, to contribute to the enrichment of the ADPKD gene database. Method In a single-center cross-sectional retrospective study, patients with renal multiple cysts diagnosed from January 2019 to February 2023 at the Zhong Da Hospital Southeast University were included. Genetic and clinical data of patients and their families were collected. Unreported novel gene mutation sites associated with ADPKD were identified, and AlphaFold v2.3.1 was employed to predict protein structures. Changes in protein structure before and after mutations were compared to explore genotype-phenotype correlations and enrich the ADPKD gene database. Results The study included 96 patients with renal multiple cysts and their relatives from 62 families. Twelve mutated genes associated with renal cysts were detected in 52 families. Nineteen novel gene mutation sites associated with ADPKD were identified (Table 1, Fig. 1) , including 17 mutations in the PKD1 gene (1 splicing mutation, 7 frameshift mutations, 4 nonsense mutations, 1 whole-codon insertion, and 4 missense mutations), 1 ALG9 missense mutation, and 1 chromosomal structural variation. Truncating mutations in the PKD1 gene were correlated with a more severe clinical phenotype, while non-truncating mutations were associated with greater clinical heterogeneity. Conclusion Numerous novel gene mutation sites associated with ADPKD remain unreported. It is essential to analyze the pathogenicity of these novel mutation sites, establish genotype-phenotype correlations, and enrich the ADPKD gene database.

#1936 Investigating genetic and environmental modifiers of autosomal dominant polycystic kidney disease through a distinct PKD2-founder variant (p.Arg803*)

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is among the most common hereditary diseases that lead to kidney failure [1]. Apart from progressive, cystic enlargement of the kidneys, classic ADPKD is inevitably associated with polycystic liver disease (PLD) and arterial hypertension [2] due to mutated PKD1 (78%) or PKD2 (15%) [3]. Despite knowledge of the diagnostic PKD1 or PKD2 variant, there is great intrafamilial variability in terms of disease progression and organ involvement. Therefore, there is an unmet medical need to establish better genotype-phenotype correlations. In 2022, Taiwanese colleagues identified a PKD2 founder variant (NM_000297.4 (PKD2): c.2407C>T (p.Arg803*)) which accounts for 17.8% of all ADPKD cases in Taiwan [4]. As we also detected patients with PKD2-Arg803* in our European center, we hypothesize that p.Arg803* constitutes a recurrent mutation. The established Taiwanese cohort provides the unique opportunity to build up a sister cohort of patients with this specific mutation. This novel approach can aid in identifying genetic and environmental modifiers of ADPKD, which in turn is essential in elucidating the pathogenesis, as well as the adequate classification, prognosis and treatment of ADPKD patients. Method For patient recruitment, the search was carried out simultaneously in a multi-pronged approach. First, the European Rare Kidney Disease Network (ERKNet), relevant registries and biobanks were searched to identify patients. In addition, authors of ClinVar entries and published literature with specific mentions of the variant were contacted. Lastly, clinical and genetic ADPKD specialists were reached out to directly. Deep phenotyping was performed by a specific clinical questionnaire that was sent to the respective treating physicians via Microsoft Forms. This included baseline information about patients as well as clinical parameters. Specifically, a detailed analysis of kidney, liver, CNS and further manifestations of ADPKD was performed. In addition to the main mutation, additional findings in relevant ADPKD genes were documented. Environmental factors such as smoking, estrogen use, diets and exercise were also been explicitly recorded. Results Through systematic search, 545 institutions or clinicians were contacted in 47 countries. Thus far, about 170 patients with PKD2-p.Arg803* have been identified in 25 centers from 15 countries outside of Taiwan. The process of deep phenotyping is ongoing (completed on 20 patients as of 01/2024). A preliminary analysis of this smaller cohort showed a mean age of 62 (range: 38–90) years and a sex distribution (F:M) of 11: 9. The median total kidney volume was 1437 ml (IQR 2525.5) mL and three patients had reached kidney failure at age 70, 72 and 69. Eighteen out of the 20 patients had three or more liver cysts with a median total liver volume of 1849 (IQR 822.5) mL. Conclusion We are continuing to expand the cohort, aiming to reach full datasets in about 80 international patients for joint analysis with the Taiwanese founder cohort. Detailed statistical analysis of the joint cohort will allow us to better characterize modifiers on the genetic and environmental level. We hope our project can contribute to further understanding disease variability in ADPKD.

#2438 ADPKD and collagen genes (COL4A3, COL4A4, COL4A5)

Abstract Background and Aims Familial hematuria diseases are a heterogeneous group of monogenic conditions caused by mutations in one of the collagen IV genes: COL4A3 (2q36.3), COL4A4 (2q36.3), and COL4A5 (Xq22.3) that are expressed in the glomerular basement membranes (GBM) and are responsible for the most frequent forms of microscopic hematuria (MH), Alport syndrome, and thin basement membrane nephropathy (TBMN). Recent data suggest that about 1% (1 in 106 individuals) of the world population may have heterozygous predicted pathogenic COL4A3 or COL4A4 variants, a frequency that leads to the occasional superimposition of TBMN with other glomerulopathies. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major genetic disorder affecting up to 12.5 million individuals worldwide and it is the fourth most common global cause of renal replacement therapy. Two are the principal causative genes: PKD1(16p13.3) and PKD2 (4q22.1). The aim of the study was to describe a cohort of clinically ADPKD patients studying collagen IV genes COL4A3, COL4A4, and COL4A5. Method We performed NGS with Sophia Genetic “Nephropathies Solution” Panel on clinically ADPKD patients (pts). This panel includes 44 genes (target region 105.8 kb) involved in different types of nephropathies. We considered variants that are categorized as Variant of Uncertain Significance (Vus), likely pathogenic, or pathogenic (Class 3,4, or 5 of American College of Medical Genetics (ACMG)) in COL4A3, COL4A4, and COL4A5 genes. Results We analyzed 250 consecutive clinically ADPKD pts (consecutive in the outpatient clinic). We found 13 (6.3%) clinically potentially interesting (Class 3,4 or 5 of ACMG) variants in collagen genes. We found 2 pts that were negative for PKD1, PKD2, PKHD1 analysis: 1 pt carrier COL4A4 NM_000092.5: c.2908C>T (p.Gln970Ter), Pathogenic and c.2756A>G (p.Glu919Gly) Vus; 1 male pt carrier COL4A3 NM_000091.5: c.609+3_609+6del, Vus and COL4A5 NM_000495:c.169G>A p.(Gly57Arg), likely pathogenic. We also found 4 pts with 4 Vus variants in collagens genes combined with at least 1 pathogenic or likely pathogenic variant in PKD1 or PKD2 genes. We found 3 pts with likely pathogenic variants in COL4A4 or COL4A3 genes combined with Vus or likely benign variants in PKD1 or PKD2 genes and 1 patient with 1 likely pathogenic variant in COL4A3 and 1 likely pathogenic variant in PKD1. Conclusion Variants in collagen genes are very frequent and the associated phenotype is varied, sometimes very mild such as microhematuria. Furthermore, penetrance has to be considered. The study outlines the importance of considering also collagen genes, even if we have a clear clinical manifestation of ADPKD. An additional variant in a kidney already compromised can have an impact on the phenotype. Analysis of genes involved in these 2 pathologies could lead to a better understanding of the phenotype-genotype correlation at least in patients with both conditions.

#787 Kidney genetics—a reproducible and curated database of kidney-disease associated genes

Abstract Background and Aims Genetic insights are becoming increasingly influential in the understanding and treatment of various kidney diseases (KD). Hundreds of genes associated with monogenic kidney disease have been identified, providing valuable insights into their diagnosis, management, and monitoring. However, the lack of a unified and standardized database of genes assigned to kidney diseases has led to diagnostic blind spots and comparability issues among current studies of kidney genetics. To address this gap, we created “Kidney-Genetics”, a regularly updated, automated and publicly accessible database, which aims to provide a comprehensive list of all relevant genes associated with kidney disease. Methods Kidney disease-associated gene information was compiled from various sources, including: (1) Genomics England and Australia PanelApp [1], (2) a comprehensive literature review of published gene lists, (3) clinical diagnostic panels for kidney disease, (4) a Human Phenotype Ontology (HPO)-based [2] search in rare disease databases (OMIM, Orphanet), and (5) a PubTator [3] API-based automated literature extraction from PubMed. An evidence-scoring system was developed to distinguish high- evidence genes from low evidence genes and candidate genes. High-evidence genes were defined as those present in two or more of the five resources and were manually curated based on predetermined criteria or, in the case of existing ClinGen [4] curation, their data and scores were used. Genes with a score of one or less were accordingly classified as candidate genes. Additionally, genes were grouped into different categories for later genotype-phenotype correlation matching (see Fig. 1). Results The Kidney-Genetics database currently contains detailed information on 3025 kidney-associated genes with detailed annotations on gene expression, kidney phenotype, inheritance mode, disease onset, possible syndromic disease mode and genetic variation. The number of genes extracted from the five analyzed sources of information are as follows: (1) 534, (2) 822, (3) 956, (4) 789, and (5) 2158 (see Fig. 2). Notably, 598 genes (19.8%) of the total 3025 genes are present in two or more of the analyzed information sources, thus meeting our evidence criteria, indicating high confidence and their potential for diagnostic use. Of these high-evidence genes, 526 (88.0%) are present in at least one, and 56 (9.4%) are present in all 10 comprehensive diagnostic laboratory panels. To ensure currency, Kidney-Genetics will be updated regularly and automatically on a monthly basis. We will also provide phenotypic and functional clustering results to facilitate gene grouping. Conclusion By utilizing Kidney-Genetics, clinicians, geneticists, and researchers can examine genomic data and improve their understanding of the genetic components of diverse KDs. The code and results are freely available on GitHub (kidney-genetics.org) [5]. A standardized pipeline and automated system keep our database on the cutting edge of kidney research and diagnostics. Screening efforts toward manual curation (such as through the ClinGen initiative) and assignment of diagnostic genes to kidney disease groups (e.g., syndromic vs. isolated; adult vs. pediatric; cystic, nephrotic, etc.) are currently under development.

Genetic heterogeneity in epilepsy and comorbidities: insights from Pakistani families

BackgroundEpilepsy, a challenging neurological condition, is often present with comorbidities that significantly impact diagnosis and management. In the Pakistani population, where financial limitations and geographical challenges hinder access to advanced diagnostic methods, understanding the genetic underpinnings of epilepsy and its associated conditions becomes crucial.MethodsThis study investigated four distinct Pakistani families, each presenting with epilepsy and a spectrum of comorbidities, using a combination of whole exome sequencing (WES) and Sanger sequencing. The epileptic patients were prescribed multiple antiseizure medications (ASMs), yet their seizures persist, indicating the challenging nature of ASM-resistant epilepsy.ResultsIdentified genetic variants contributed to a diverse range of clinical phenotypes. In the family 1, which presented with epilepsy, developmental delay (DD), sleep disturbance, and aggressive behavior, a homozygous splice site variant, c.1339–6 C > T, in the COL18A1 gene was detected. The family 2 exhibited epilepsy, intellectual disability (ID), DD, and anxiety phenotypes, a homozygous missense variant, c.344T > A (p. Val115Glu), in the UFSP2 gene was identified. In family 3, which displayed epilepsy, ataxia, ID, DD, and speech impediment, a novel homozygous frameshift variant, c.1926_1941del (p. Tyr643MetfsX2), in the ZFYVE26 gene was found. Lastly, family 4 was presented with epilepsy, ID, DD, deafness, drooling, speech impediment, hypotonia, and a weak cry. A homozygous missense variant, c.1208 C > A (p. Ala403Glu), in the ATP13A2 gene was identified.ConclusionThis study highlights the genetic heterogeneity in ASM-resistant epilepsy and comorbidities among Pakistani families, emphasizing the importance of genotype-phenotype correlation and the necessity for expanded genetic testing in complex clinical cases.

Biochemical and molecular analysis of pediatric patients with metachromatic leukodystrophy in South China: functional characterization of five novel ARSA variants.

Metachromatic leukodystrophy (MLD) is a rare hereditary neurodegenerative disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). This study described the clinical and molecular characteristics of 24 Chinese children with MLD and investigated functional characterization of five novel ARSA variants. A retrospective analysis was performed in 24 patients diagnosed with MLD at Guangzhou Women and Children's Medical Center in South China. Five novel mutations were further characterized by transient expression studies. We recruited 17 late-infantile, 3 early-juvenile, 4 late-juvenile MLD patients. In late-infantile patients, motor developmental delay and gait disturbance were the most frequent symptoms at onset. In juvenile patients, cognitive regression and gait disturbance were the most frequent chief complaints. Overall, 25 different ARSA mutations were identified with 5 novel mutations.The most frequent alleles were p.W320* and p.G449Rfs. The mutation p.W320*, p.Q155=, p.P91L, p.G156D, p.H208Mfs*46 and p.G449Rfs may link to late-infantile type. The novel missense mutations were predicted damaging in silico. The bioinformatic structural analysis of the novel missense mutations showed that these amino acid replacements would cause severe impairment of protein structure and function. In vitro functional analysis of the six mutants, showing a low ARSA enzyme activity, clearly demonstrated their pathogenic nature. The mutation p.D413N linked to R alleles. In western blotting analysis of the ARSA protein, the examined mutations retained reduced amounts of ARSA protein compared to the wild type. This study expands the spectrum of genotype of MLD. It helps to the future studies of genotype-phenotype correlations to estimate prognosis and develop new therapeutic approach.

Exome sequencing in four families with neurodevelopmental disorders: genotype-phenotype correlation and identification of novel disease-causing variants in VPS13B and RELN.

Neurodevelopmental disorders (NDDs) are a clinically and genetically heterogeneous group of early-onset pediatric disorders that affect the structure and/or function of the central or peripheral nervous system. Achieving a precise molecular diagnosis for NDDs may be challenging due to the diverse genetic underpinnings and clinical variability. In the current study, we investigated the underlying genetic cause(s) of NDDs in four unrelated Pakistani families. Using exome sequencing (ES) as a diagnostic approach, we identified disease-causing variants in established NDD-associated genes in all families, including one hitherto unreported variant in RELN and three recurrent variants in VPS13B, DEGS1, and SPG11. Overall, our study highlights the potential of ES as a tool for clinical diagnosis.

Complex genotype–phenotype correlation of MYH11: new insights from monozygotic twins with highly variable expressivity and outcomes

BackgroundThoracic 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.MethodsThe 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.ResultsThe 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).ConclusionThis 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.

Genotype-phenotype correlations of AR-CMT2S in a cohort of axonal Charcot-Marie-Tooth patients from Central South China.

This study aimed to report nine Charcot-Marie-Tooth disease (CMT) families with six novel IGHMBP2 mutations in our CMT2 cohort and to summarize the genetic and clinical features of all AR-CMT2S patients reported worldwide. General information, clinical and neurophysiological data of 275 axonal CMT families were collected. Genetic screening was performed by inherited peripheral neuropathy related genes panel or whole exome sequencing. The published papers reporting AR-CMT2S from 2014 to 2023 were searched in Pubmed and Wanfang databases. In our CMT2 cohort, we detected 17 AR-CMT2S families carrying IGHMBP2 mutations and eight were published previously. Among these, c.743 T > A (p.Val248Glu), c.884A > G (p.Asp295Gly), c.1256C > A (p.Ser419*), c.2598_2599delGA (p.Lys868Sfs*16), c.1694_1696delATG (p.Asp565del) and c.2509A > T (p.Arg837*) were firstly reported. These patients prominently presented with early-onset typical axonal neuropathy and without respiratory dysfunction. So far, 56 AR-CMT2S patients and 57 different mutations coming from 43 families have been reported in the world. Twenty-nine of 32 missense mutations were clustered in helicase domain and ATPase region. The age at onset ranged from 0.11to 20 years (Mean ± SD: 3.43 ± 3.88 years) and the majority was infantile-onset (<2 years). The initial symptoms included weakness of limbs (19, 29.7%), delayed milestones (12, 18.8%), gait disturbance (11, 17.2%), feet deformity (8, 12.5%), feet drop (8, 12.5%), etc. INTERPRETATION: AR-CMT2S accounted for 6.2% in our CMT2 cohort. We firstly reported six novel IGHMBP2 mutations which expanded the genotypic spectrum of AR-CMT2S. Furthermore, 17 AR-CMT2S families could provide more resources for natural history study, drug research and development.

A Novel Transmembrane CXCR4 Variant That Expands the WHIM Genotype-Phenotype Paradigm

Warts, Hypogammglobulinemia, Infections, Myelokathexis (WHIM) syndrome is a rare immunodeficiency disease that results from impaired leukocyte trafficking (myelokathexis) predominately caused by gain-of-function variants in C-X-C chemokine receptor type 4 (CXCR4). Clinical manifestations of WHIM syndrome can differ in familial forms or in people harboring identical CXCR4 variants. All known pathogenic CXCR4 variants associated with WHIM syndrome (CXCR4WHIM) to date are localized in the intracellular C-terminus of CXCR4. We identified 4 unrelated patients with variable WHIM-like clinical presentations harboring a novel heterozygous CXCR4 variant (c.250G>C; p.D84H) localized at a highly conserved position in the transmembrane domain of the receptor outside the C-terminus. Functional characterization of the CXCR4D84Hvariant (CXCR4D84H) using patient-derived peripheral blood mononuclear cells and in vitro cellular assaysshow decreased CXCR4 internalization and increased chemotaxis in response to CXCL12, similar to known CXCR4WHIM, but also revealed unique features of CXCR4D84H signaling to cAMP, Ca2+ mobilization and AKT/ERK pathways. These findings are consistent with molecular dynamics simulations that show disruption of the Na+ binding pocket by D84H, resulting in collapse of the hydrophobic gate above and destabilization of the inactive state of CXCR4. Mavorixafor, a CXCR4 antagonist being evaluated in clinical trials for chronic neutropenia and WHIM syndrome, normalized CXCL12-mediated chemotaxis of CXCR4D84H patient lymphocytes ex vivo and improved WBC and subset counts in 1 patient with CXCR4D84H enrolled in the chronic neutropenia phase 1b clinical trial (NCT04154488). The present study expands the current understanding of CXCR4 function and genotype-phenotype correlations in WHIM syndrome and in people with WHIM-like phenotypes.

Improving the Yield of Genetic Diagnosis through Additional Genetic Panel Testing in Hereditary Ophthalmic Diseases.

Numerous hereditary ophthalmic diseases display significant genetic diversity. Consequently, the utilization of gene panel sequencing allows a greater number of patients to receive a genetic diagnosis for their clinical manifestations. We investigated how to improve the yield of genetic diagnosis through additional gene panel sequencing in hereditary ophthalmic diseases. A gene panel sequencing consisting of a customized hereditary retinopathy panel or hereditary retinitis pigmentosa (RP) panel was prescribed and referred to a CAP-accredited clinical laboratory. If no significant mutations associated with hereditary retinopathy and RP were detected in either panel, additional gene panel sequencing was requested for research use, utilizing the remaining panel. After additional gene panel sequencing, a total of 16 heterozygous or homozygous variants were identified in 15 different genes associated with hereditary ophthalmic diseases. Of 15 patients carrying any candidate variants, the clinical symptoms could be tentatively accounted for by genetic mutations in seven patients. However, in the remaining eight patients, given the in silico mutation predictive analysis, variant allele frequency in gnomAD, inheritance pattern, and genotype-phenotype correlation, fully elucidating the clinical manifestations with the identified rare variant was challenging. Our study highlights the utility of gene panel sequencing in achieving accurate diagnoses for hereditary ophthalmic diseases and enhancing the diagnostic yield through additional gene panel sequencing. Thus, gene panel sequencing can serve as a primary tool for the genetic diagnosis of hereditary ophthalmic diseases, even in cases where a single genetic cause is suspected. With a deeper comprehension of the genetic mechanisms underlying these diseases, it becomes feasible.

Phenotypic Variability in Novel Doublecortin Gene Variants Associated with Subcortical Band Heterotopia.

Doublecortin, encoded by the DCX gene, plays a crucial role in the neuronal migration process during brain development. Pathogenic variants of the DCX gene are the major causes of the "lissencephaly (LIS) spectrum", which comprehends a milder phenotype like Subcortical Band Heterotopia (SBH) in heterozygous female subjects. We performed targeted sequencing in three unrelated female cases with SBH. We identified three DCX-related variants: a novel missense (c.601A>G: p.Lys201Glu), a novel nonsense (c.210C>G: p.Tyr70*), and a previously identified nonsense (c.907C>T: p.Arg303*) variant. The novel c.601A>G: p.Lys201Glu variant shows a mother-daughter transmission pattern across four generations. The proband exhibits focal epilepsy and achieved seizure freedom with a combination of oxcarbazepine and levetiracetam. All other affected members have no history of epileptic seizures. Brain MRIs of the affected members shows predominant fronto-central SBH with mixed pachygyria on the overlying cortex. The two nonsense variants were identified in two unrelated probands with SBH, severe drug-resistant epilepsy and intellectual disability. These novel DCX variants further expand the genotypic-phenotypic correlations of lissencephaly spectrum disorders. Our documented phenotypic descriptions of three unrelated families provide valuable insights and stimulate further discussions on DCX-SBH cases.

Molecular and Clinical Features of Congenital Hypothyroidism due to Multiple DUOX2 Variants.

Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown function. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in the clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutation spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.