A novel homozygous splice site variant in ARL2BP causes a syndromic autosomal recessive rod-cone dystrophy with situs inversus, asthenozoospermia, unilateral renal agenesis and microcysts.

BACKGROUND: This report presents a clinical case of syndromic rod-cone dystrophy due to a splice site variant in the ARL2BP gene causing situs inversus, asthenozoospermia, unilateral renal agenesis and microcysts. The presence of renal agenesis and cryptorchidism expands the clinical manifestations due to ARL2BP variants. The detailed, long-term follow-up contributes valuable insights into disease progression, aiding clinical diagnosis and patient management. CASE PRESENTATION: The male patient complained of photophobia as the first symptom when he was 20 years old followed by nyctalopia, loss of central visual acuity and peripheral visual field ten years later. Genetic analysis identified a likely pathogenic homozygous variant (c.294-1G > C) involving the splicing acceptor site of intron 4. Reported symptoms together with full-field stimulus threshold testing, electroretinogram and advanced multimodal imaging allowed us to recognize the typical characteristics of a mixed retinal dystrophy. Despite the end-stage retinal disease, this patient still retained a useful residual vision at 63 years and had a slow disease progression during the last 5 years of evaluation. DISCUSSION AND CONCLUSIONS: Our findings underscore the variable clinical presentation of ARL2BP variants, emphasizing the importance of a nuanced approach in diagnosing and managing patients. The presence of renal cysts warrants consideration of a differential diagnosis, particularly with Senior-Loken (SLS), Bardet-Biedl (BBS) and Joubert syndromes (JS) but also with Short Rib Thoracic Dysplasia 9, highlighting the need for careful phenotypic evaluation in these cases.

Prenatal CFAP53-related laterality defect: case report and review of the literature.

Laterality defects include morphological anomalies with impaired left-right asymmetry induction, such as dextrocardia, situs inversus abdominis, situs inversus totalis and situs ambiguus. The different arrangement of major organs is called heterotaxy. We describe for the first time a fetus with situs viscerum inversus and azygos continuation of the inferior vena cava, due to previously unreported variants in compound heterozygosity in the CFAP53 gene, whose product is implied in cilial motility. Prenatal trio exome sequencing was performed with turn-around time during the pregnancy. The fetuses with laterality defects are suitable candidates for prenatal exome sequencing due to the emerging high diagnostic rate of this group of morphological anomalies. A timely molecular diagnosis plays a fundamental role in genetic counseling, regarding couple decisions on the ongoing pregnancy, providing recurrence risks, and in predicting possible respiratory complications due to ciliary dyskinesia.

Novel homozygous variant in ARL2BP associated with retinitis pigmentosa, situs inversus, and male infertility in a Chinese patient.

ARL2BP is a ciliary gene associated with multiple ciliopathy phenotypes. On comprehensive clinical examinations using molecular methods, we identified a Chinese patient from a consanguineous family carrying a novel homozygous variant c.22_23delAG (p.S8Lfs*10) in ARL2BP, presenting with retinitis pigmentosa (RP), situs inversus totalis, and oligozoospermia. Situs inversus and male infertility have never been reported in the same patient with ARL2BP variants; therefore, this a novel ARL2BP-associated phenotypic triad of RP, situs inversus, and male infertility. Moreover, this patient likely had olfactory dysfunction susceptibility and presented with anosmia. We found reduced patient-derived fibroblast proliferation and ciliary length. Our findings expand the genotypic spectrum and reveal abnormal cell proliferation and ciliogenesis in ARL2BP-associated patients.

A novel nonsense PKD1L1 variant cause heterotaxy syndrome with congenital asplenia in a Han Chinese patient.

Heterotaxy syndrome is a very rare congenital disease, which is caused by the disorder of left-right asymmetry during visceral development. However, pathogenic genetic lesions are found in less than 20% of HS patients. In this cohort study, whole-exome sequencing was performed for 110 patients with situs inversus or situs ambiguous. We identified a novel nonsense variant in PKD1L1(c.1387 C > T; p.463Gln*) in a Chinese patient with heterotaxy syndrome and congenital asplenia. This homozygous variant caused the domain of PKD1L1 complete absence. To our knowledge, this novel variant is the first phenotype of congenital asplenia found in patients with PKD1L1 variants, and the first PKD1L1 variant found in China. Our findings expand the spectrum of PKD1L1 variants and provide support for PKD1L1 variant and congenital asplenia, and the critical role of PKD1L1 during left-right patterning in the Han Chinese population.

Exome sequencing in individuals with cardiovascular laterality defects identifies potential candidate genes.

The birth prevalence of laterality defects is about 1.1/10,000 comprising different phenotypes ranging from situs inversus totalis to heterotaxy, mostly associated with complex congenital heart defects (CHD) and situs abnormalities such as intestinal malrotation, biliary atresia, asplenia, or polysplenia. A proportion of laterality defects arise in the context of primary ciliary dyskinesia (PCD) accompanied by respiratory symptoms or infertility. In this study, exome sequencing (ES) was performed in 14 case-parent trios/quattros with clinical exclusion of PCD prior to analysis. Moreover, all cases and parents underwent detailed clinical phenotyping including physical examination, echocardiography by a skilled paediatric cardiologist and abdominal ultrasound examinations not to miss mildly affected individuals. Subsequent survey of the exome data comprised filtering for monoallelic de novo, rare biallelic, and X-linked recessive variants. In two families, rare variants of uncertain significance (VUS) in PKD1L1 and ZIC3 were identified. Both genes have been associated with laterality defects. In two of the remaining families, biallelic variants in LMBRD1 and DNAH17, respectively, were prioritized. In another family, an ultra-rare de novo variant in WDR47 was found. Extensive exome survey of 2,109 single exomes of individuals with situs inversus totalis, heterotaxy, or isolated CHD identified two individuals with novel monoallelic variants in WDR47, but no further individuals with biallelic variants in DNAH17 or LMBRD1. Overall, ES of 14 case-parent trios/quattros with cardiovascular laterality defects identified rare VUS in two families in known disease-associated genes PKD1L1 and ZIC3 and suggests DNAH17, LMBRD1, and WDR47 as potential genes involved in laterality defects.

Compound heterozygous variants in DYNC2H1 in a foetus with type III short rib-polydactyly syndrome and situs inversus totalis.

BACKGROUND: Short-rib thoracic dysplasia 3 with or without polydactyly (SRTD3, OMIM: 613091) is an autosomal recessive disorder. SRTD3 presents clinically with a narrow thorax, short ribs, shortened tubular bones, and acetabular roof abnormalities. Clinical signs of SRTD3 vary among individuals. Pathogenic variants of DYNC2H1 (OMIM: 603297) have been reported to cause SRTD3. METHODS: We performed a detailed clinical prenatal sonographic characterization of a foetus with SRTD3. Trio whole-exome sequencing was used to identify causative variants in the family. The identified variants in the families were validated by Sanger sequencing and mass spectrometry. Multiple computational tools were used to predict the harmfulness of the two variants. A minigene splicing assay was carried out to evaluate the impact of the splice-site variant. RESULTS: We evaluated prenatal sonographic images of the foetus with SRTD3, including abnormal rib curvature, narrow thorax, bilateral hypoplastic lungs, bilateral polydactyly, syndactyly, and foetal visceral situs inversus with mirror-image dextrocardia. We revealed novel compound variants of DYNC2H1 (NM_001377.3:c.11483T > G (p.Ile3828Arg) and c.2106 + 3A > T). Various statistical methods predicted that the variants would cause harmful effects on genes or gene products. The minigene assay findings suggested that c.2106 + 3A > T caused the skipping over exon 14, producing an exon 14 loss in the protein. CONCLUSION: This study identified a foetus with SRTD3 with situs inversus totalis with mirror-image dextrocardia in a Chinese family, revealing two novel compound heterozygous dynein cytoplasmic 2 heavy chain 1 (DYNC2H1) variants, expanding the phenotypic spectrum of SRTD3. The minigene study of c.2106 + 3A > T was predicted to cause an inframe exclusion of exon 14, which was predicted to have important molecular functions. Our findings strongly supported the use of WES in prenatal diagnosis and helped to understand the correlation of genotype and phenotypes of DYNC2H1. The specific sonographic findings and the molecular diagnosis helped add experience to further our expertise in prenatal counselling for SRTD3.

Biallelic DNAH9 mutations are identified in Chinese patients with defective left-right patterning and cilia-related complex congenital heart disease.

Defective left-right (LR) pattering results in a spectrum of laterality disorders including situs inversus totalis (SIT) and heterotaxy syndrome (Htx). Approximately, 50% of patients with primary ciliary dyskinesia (PCD) displayed SIT. Recessive variants in DNAH9 have recently been implicated in patients with situs inversus. Here, we describe six unrelated family trios and 2 sporadic patients with laterality defects and complex congenital heart disease (CHD). Through whole exome sequencing (WES), we identified compound heterozygous mutations in DNAH9 in the affected individuals of these family trios. Ex vivo cDNA amplification revealed that DNAH9 mRNA expression was significantly downregulated in these patients carrying biallelic DNAH9 mutations, which cause a premature stop codon or exon skipping. Transmission electron microscopy (TEM) analysis identified ultrastructural defects of the outer dynein arms in these affected individuals. dnah9 knockdown in zebrafish lead to the disturbance of cardiac left-right patterning without affecting ciliogenesis in Kupffer's vesicle (KV). By generating a Dnah9 knockout (KO) C57BL/6n mouse model, we found that Dnah9 loss leads to compromised cardiac function. In this study, we identified recessive DNAH9 mutations in Chinese patients with cardiac abnormalities and defective LR pattering.

[Dyspnea and situs inversus in a boy aged 3 days].

A boy was admitted on day 3 after birth due to shortness of breath for 2 days and cyanosis for 1 day. He had clinical manifestations of dyspnea in the early postnatal period and situs inversus, and was finally diagnosed with Kartagener syndrome. His condition was improved after oxygen therapy, anti-infective therapy, and aerosol therapy. The genetic testing showed that there was a large-fragment loss of heterozygosity, exon 48_50, and a hemizygous mutation, c.7915C > T(p.R2639X), in the DNAH5 gene. Kartagener syndrome is a rare autosomal recessive disease, and this is the first case of Kartagener syndrome diagnosed in the neonatal period in China.

Rare association of Klippel-Feil syndrome with situs inversus totalis and review of the genetic background.

Klippel-Feil syndrome (KFS) is a rare congenital anomaly in forming the cervical vertebrae resulting in the fusion of two or more of the vertebrae. KFS is associated with many congenital anomalies, some of which are common and well known. Here, we report a child with an extremely rare association of KFS with situs inversus totalis (SIT). Both KFS and SIT are genetically heterogeneous and their co-occurrence suggests a high possibility of sharing the same underlying causative agent. Here, we review the genetic background that is known for these two conditions in the literature.

Whole-exome sequencing reveals a combination of extremely rare single-nucleotide polymorphism of DNAH9 and RSPH1 genes in a Japanese fetus with situs viscerum inversus.

Randomization of left-right body asymmetry, situs viscerum inversus (heterotaxy), is commonly associated with primary ciliary dyskinesia (PCD) resulting from an abnormal ciliary structure, with approximately 50% of PCD patients exhibiting organ laterality defects. I herein report an intrauterine fetal death case, in which an autopsy revealed two lobes of the bilateral lungs as well as heterotaxy of abdominal organs (right-sided spleen and inversion of the alimentary and biliary organs). Whole-exome sequencing (WES) identified a heterozygous single-nucleotide change (c.12775T>C) in exon 68 of the DNAH9 gene, which is a rare single-nucleotide polymorphism (SNP) of rs746081639 and results in the amino acid change of p.C4259R. WES also identified a rare SNP of rs763089682 (c.121G>A) in the RSPH1 gene that causes a heterozygous amino acid alteration of p.G41R. The frequencies of both SNPs, C in rs746081639 and A in rs763089682, are 0.00000824, and a polyphen-2 analysis predicted these amino acid changes to be probably damaging, with a score of 1.000. The combination of extremely rare SNPs in DNAH9 and RSPH1 genes might have been the possible mechanism underlying the development of the laterality defect in the present case.

Hydrops fetalis in PKD1L1-related heterotaxy: Report of two foetuses and expanding the phenotypic and molecular spectrum.

Abnormalities in the normal left-right axis asymmetry range from situs inversus totalis to situs ambiguous or heterotaxy. More than 80 genes have been described to have a role in the establishment of the normal situs of the internal organs. Pathogenic variants in the PKD1L1 gene have recently been described in heterotaxy and congenital heart disease. Till date, 11 families have been described with PKD1L1-related heterotaxy. We describe the first Indian family with two affected foetuses with PKD1L1-related nonimmune hydrops, congenital heart disease, situs inversus, and heterotaxy, with biallelic variants in the compound heterozygous state.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Rare variants in dynein heavy chain genes in two individuals with situs inversus and developmental dyslexia: a case report.

BACKGROUND: Developmental dyslexia (DD) is a neurodevelopmental learning disorder with high heritability. A number of candidate susceptibility genes have been identified, some of which are linked to the function of the cilium, an organelle regulating left-right asymmetry development in the embryo. Furthermore, it has been suggested that disrupted left-right asymmetry of the brain may play a role in neurodevelopmental disorders such as DD. However, it is unknown whether there is a common genetic cause to DD and laterality defects or ciliopathies. CASE PRESENTATION: Here, we studied two individuals with co-occurring situs inversus (SI) and DD using whole genome sequencing to identify genetic variants of importance for DD and SI. Individual 1 had primary ciliary dyskinesia (PCD), a rare, autosomal recessive disorder with oto-sino-pulmonary phenotype and SI. We identified two rare nonsynonymous variants in the dynein axonemal heavy chain 5 gene (DNAH5): a previously reported variant c.7502G > C; p.(R2501P), and a novel variant c.12043 T > G; p.(Y4015D). Both variants are predicted to be damaging. Ultrastructural analysis of the cilia revealed a lack of outer dynein arms and normal inner dynein arms. MRI of the brain revealed no significant abnormalities. Individual 2 had non-syndromic SI and DD. In individual 2, one rare variant (c.9110A > G;p.(H3037R)) in the dynein axonemal heavy chain 11 gene (DNAH11), coding for another component of the outer dynein arm, was identified. CONCLUSIONS: We identified the likely genetic cause of SI and PCD in one individual, and a possibly significant heterozygosity in the other, both involving dynein genes. Given the present evidence, it is unclear if the identified variants also predispose to DD and further studies into the association between laterality, ciliopathies and DD are needed.

The genetics of situs inversus without primary ciliary dyskinesia.

Situs inversus (SI), a left-right mirror reversal of the visceral organs, can occur with recessive Primary Ciliary Dyskinesia (PCD). However, most people with SI do not have PCD, and the etiology of their condition remains poorly studied. We sequenced the genomes of 15 people with SI, of which six had PCD, as well as 15 controls. Subjects with non-PCD SI in this sample had an elevated rate of left-handedness (five out of nine), which suggested possible developmental mechanisms linking brain and body laterality. The six SI subjects with PCD all had likely recessive mutations in genes already known to cause PCD. Two non-PCD SI cases also had recessive mutations in known PCD genes, suggesting reduced penetrance for PCD in some SI cases. One non-PCD SI case had recessive mutations in PKD1L1, and another in CFAP52 (also known as WDR16). Both of these genes have previously been linked to SI without PCD. However, five of the nine non-PCD SI cases, including three of the left-handers in this dataset, had no obvious monogenic basis for their condition. Environmental influences, or possible random effects in early development, must be considered.

SPEF2- and HYDIN-Mutant Cilia Lack the Central Pair-associated Protein SPEF2, Aiding Primary Ciliary Dyskinesia Diagnostics.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2, which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN-mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus. Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.

MNS1 variant associated with situs inversus and male infertility.

Ciliopathy disorders due to abnormalities of motile cilia encompass a range of autosomal recessive conditions typified by chronic otosinopulmonary disease, infertility, situs abnormalities and hydrocephalus. Using a combination of genome-wide SNP mapping and whole exome sequencing (WES), we investigated the genetic cause of a form of situs inversus (SI) and male infertility present in multiple individuals in an extended Amish family, assuming that an autosomal recessive founder variant was responsible. This identified a single shared (2.34 Mb) region of autozygosity on chromosome 15q21.3 as the likely disease locus, in which we identified a single candidate biallelic frameshift variant in MNS1 [NM_018365.2: c.407_410del; p.(Glu136Glyfs*16)]. Genotyping of multiple family members identified randomisation of the laterality defects in other homozygous individuals, with all wild type or MNS1 c.407_410del heterozygous carriers being unaffected, consistent with an autosomal recessive mode of inheritance. This study identifies an MNS1 variant as a cause of laterality defects and male infertility in humans, mirroring findings in Mns1-deficient mice which also display male infertility and randomisation of left-right asymmetry of internal organs, confirming a crucial role for MNS1 in nodal cilia and sperm flagella formation and function.

Characterization of CCDC103 expression profiles: further insights in primary ciliary dyskinesia and in human reproduction.

PROPOSE: To study CCDC103 expression profiles and understand how pathogenic variants in CCDC103 affect its expression profile at mRNA and protein level. METHODS: To increase the knowledge about the CCDC103, we attempted genotype-phenotype correlations in two patients carrying novel homozygous (missense and frameshift) CCDC103 variants. Whole-exome sequencing, quantitative PCR, Western blot, electron microscopy, immunohistochemistry, immunocytochemistry, and immunogold labelling were performed to characterize CCDC103 expression profiles in reproductive and somatic cells. RESULTS: Our data demonstrate that pathogenic variants in CCDC103 gene negatively affect gene and protein expression in both patients who presented absence of DA on their axonemes. Further, we firstly report that CCDC103 is expressed at different levels in reproductive tissues and somatic cells and described that CCDC103 protein forms oligomers with tissue-specific sizes, which suggests that CCDC103 possibly undergoes post-translational modifications. Moreover, we reported that CCDC103 was restricted to the midpiece of sperm and is present at the cytoplasm of the other cells. CONCLUSIONS: Overall, our data support the CCDC103 involvement in PCD and suggest that CCDC103 may have different assemblies and roles in cilia and sperm flagella biology that are still unexplored.

Nitric Oxide Reverses the Position of the Heart during Embryonic Development.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays crucial roles in cardiac homeostasis. Adult cardiomyocyte specific overexpression of eNOS confers protection against myocardial-reperfusion injury. However, the global effects of NO overexpression in developing cardiovascular system is still unclear. We hypothesized that nitric oxide overexpression affects the early migration of cardiac progenitor cells, vasculogenesis and function in a chick embryo. Vehicle or nitric oxide donor DEAN (500 mM) were loaded exogenously through a small window on the broad side of freshly laid egg and embryonic development tracked by live video-microscopy. At Hamburg Hamilton (HH) stage 8, the cardiac progenitor cells (CPC) were isolated and cell migration analysed by Boyden Chamber. The vascular bed structure and heart beats were compared between vehicle and DEAN treated embryos. Finally, expression of developmental markers such as BMP4, Shh, Pitx2, Noggin were measured using reverse transcriptase PCR and in-situ hybridization. The results unexpectedly showed that exogenous addition of pharmacological NO between HH stage 7⁻8 resulted in embryos with situs inversus in 28 out of 100 embryos tested. Embryos treated with NO inhibitor cPTIO did not have situs inversus, however 10 embryos treated with L-arginine showed a situs inversus phenotype. N-acetyl cysteine addition in the presence of NO failed to rescue situs inversus phenotype. The heart beat is normal (120 beats/min) although the vascular bed pattern is altered. Migration of CPCs in DEAN treated embryos is reduced by 60% compared to vehicle. BMP4 protein expression increases on the left side of the embryo compared to vehicle control. The data suggests that the NO levels in the yolk are important in turning of the heart during embryonic development. High levels of NO may lead to situs inversus condition in avian embryo by impairing cardiac progenitor cell migration through the NO-BMP4-cGMP axis.