A Japanese patient with Teebi hypertelorism syndrome and a novel CDH11 EC1 domain variant.

The gene CDH11 encodes cadherin-11, a Type II cadherin superfamily member that contains five extracellular cadherin (EC) domains. Cadherin-11 undergoes trans-dimerization via the EC1 domain to generate cadherin complexes. Compound heterozygous and homozygous loss-of-function CDH11 variants are observed in Elsahy-Waters syndrome (EWS), which shows characteristic craniofacial features, vertebral abnormalities, cutaneous syndactyly in 2-3 digits, genitourinary anomalies, and intellectual disability. Heterozygous CDH11 variants can cause Teebi hypertelorism syndrome (THS), which features widely spaced eyes and hypospadias. We report a THS patient with a novel CDH11 variant involving the EC1 domain. The patient was a 10-month-old male with normal developmental milestones, but had widely spaced eyes, strabismus, hypospadias, shawl scrotum, broad thumbs (right bifid thumb in x-ray), polysyndactyly of the left fourth finger, and cutaneous syndactyly of left third/fourth fingers. Exome sequencing identified a de novo heterozygous CDH11 variant (NM_001797.4:c.229C > T [p.Leu77Phe] NC_000016.9:g.64998856G > A). Clinical features were consistent with previously reported THS patients, but polysyndactyly, broad thumb, and cutaneous syndactyly overlapped phenotypic features of EWS. THS and EWS may represent a spectrum of CDH11-related disorders. Residue Leu77 in this novel CDH11 variant lines a large hydrophobic pocket where side chains of the partner cadherin-11 insert to trans-dimerize, suggesting that the cadherin-11 structure might be altered in this variant.

Opitz GBBB syndrome with total anomalous pulmonary venous connection: A new MID1 gene variant.

BACKGROUND: Opitz GBBB syndrome (GBBB) is an X-linked disease characterized by midline defects, including congenital heart defects. We present our diagnostic approach to the identification of GBBB in a consanguineous family in which two males siblings were concordant for a total anomalous connection of pulmonary veins and minor facial dysmorphias. METHODS: Targeted exome sequencing analysis of a 380-gene panel associated with cardiovascular disease was performed on the propositus. Interpretative analysis of the exome results was conducted, and 3D models of the protein changes were generated. RESULTS: We identified a NM_000381.4:c.608G>A;p.(Arg203Gln) change in MID1, affecting the conformation of the B-box 2 domain of the protein, with a zinc finger structure and associated protein interactions. This clinical phenotype is consistent with GBBB; however, the type of congenital heart disease observed in this case has not been previously reported. CONCLUSION: A new likely pathogenic variant on MID1 c.608G>A was found to be associated with Opitz GBBB syndrome.

Opitz syndrome: improving clinical interpretation of intronic variants in MID1 gene.

BACKGROUND: Loss-of-function variants in MID1 are the most common cause of Opitz G/BBB syndrome (OS). The interpretation of intronic variants affecting the splicing is a rising issue in OS. METHODS: Exon sequencing of a 2-year-old boy with OS showed that he was a carrier of the de novo c.1286-10G>T variant in MID1. In silico predictions and minigene assays explored the effect of the variant on splicing. The minigene approach was also applied to two previously identified MID1 c.864+1G>T and c.1285+1G>T variants. RESULTS: Minigene assay demonstrated that the c.1286-10G>T variant generated the inclusion of eight nucleotides that predicted generation of a frameshift. The c.864+1G>T and c.1285+1G>T variants resulted in an in-frame deletion predicted to generate a shorter MID1 protein. In hemizygous males, this allowed reclassification of all the identified variants from "of unknown significance" to "likely pathogenic." CONCLUSIONS: Minigene assay supports functional effects from MID1 intronic variants. This paves the way to the introduction of similar second-tier investigations in the molecular diagnostics workflow of OS. IMPACT: Causative intronic variants in MID1 are rarely investigated in Opitz syndrome. MID1 is not expressed in blood and mRNA studies are hardly accessible in routine diagnostics. Minigene assay is an alternative for assessing the effect of intronic variants on splicing. This is the first study characterizing the molecular consequences of three MID1 variants for diagnostic purposes and demonstrating the efficacy of minigene assays in supporting their clinical interpretation. Review of the criteria according to the American College of Medical Genetics reassessed all variants as likely pathogenic.

SPECC1L Mutations Are Not Common in Sporadic Cases of Opitz G/BBB Syndrome.

Opitz G/BBB syndrome (OS) is a rare genetic developmental condition characterized by congenital defects along the midline of the body. The main clinical signs are represented by hypertelorism, laryngo-tracheo-esophageal defects and hypospadias. The X-linked form of the disease is associated with mutations in the MID1 gene located in Xp22 whereas mutations in the SPECC1L gene in 22q11 have been linked to few cases of the autosomal dominant form of this disorder, as well as to other genetic syndromes. In this study, we have undertaken a mutation screening of the SPECC1L gene in samples of sporadic OS cases in which mutations in the MID1 gene were excluded. The heterozygous missense variants identified are already reported in variant databases raising the issue of their pathogenetic meaning. Recently, it was reported that some clinical manifestations peculiar to OS signs are not observed in patients carrying mutations in the SPECC1L gene, leading to the proposal of the designation of 'SPECC1L syndrome' to refer to this disorder. Our study confirms that patients with diagnosis of OS, mainly characterized by the presence of hypospadias and laryngo-tracheo-esophageal defects, do not carry pathogenic SPECC1L mutations. In addition, SPECC1L syndrome-associated mutations are clustered in two specific domains of the protein, whereas the missense variants detected in our work lies elsewhere and the impact of these variants in the function of this protein is difficult to ascertain with the current knowledge and will require further investigations. Nonetheless, our study provides further insight into the SPECC1L syndrome classification.

Pseudo-Roberts Syndrome: An Entity or Not?

Background: Roberts syndrome is a genetic disorder characterized by tetra-phocomelia with abnormalities of ESCO2. We report a male stillborn with tetra-phocomelia and no ESCO2 mutation. Case report: Pre- and post-natal imaging and autopsy findings included schizencephaly, phocomelia of four limbs, micrognathia, oligodactyly, and cardiopulmonary malformations. Microcephaly on pre-natal imaging was not confirmed by autopsy examination. Karyotype, prenatal chromosome microarray and ESCO2 gene testing were normal. Conclusion: Given the various skeletal anomalies found on autopsy and imaging evaluations, at least phenotypically, our case appeared to conform into Roberts syndrome spectrum. Since the infant did not have the mutation associated with this disorder, this infant could be labeled as the first report of a pseudo-Roberts syndrome because many of his phenotypic anomalies are characteristic of Roberts syndrome in absence of the ESCO2 gene mutation.

ALX-Related Frontonasal Dysplasias: Clinical Characteristics and Surgical Management.

AIM: The term frontonasal dysplasia (FND) represents a spectrum of anomalies and its genetics have not been well defined. Recently, the critical role of the aristaless-like homeobox (ALX) gene family on the craniofacial development has been discovered. In the present study, we aimed to propose a systematic surgical treatment plan for the ALX-related FNDs according to the genotypic classification as well as demonstrating their clinical characteristics to help surgeons diagnose the underlying pathology accurately. DESIGN: Single-institution retrospective. SETTING: Tertiary health care. PATIENTS AND METHODS: Eighty-nine FND cases were evaluated. Eight of them had ALX1-related FND3, 3 had ALX3-related FND1, and 2 had ALX4-related FND2. Phenotype characteristics of ALX-related FNDs were evaluated, and relevant surgical interventions were assessed. RESULTS: The ALX1-related FND3 phenotype is striking due to the involvement of the eyes in addition to the presence of hypertelorism, facial clefts, and nasal deformities. A widened philtrum and prominent philtral columns are remarkable features of the ALX3-related FND1, whereas the ALX4-related FND2 has more severe deformities: severe hypertelorism, brachycephaly, large parietal bone defects, broad nasal dorsum, and alopecia. Facial bipartition, box osteotomies, eyelid coloboma repair, cleft lip and palate repair, nasal reconstruction, and fronto-orbital advancement can be performed in ALX-related FNDs based on the characteristics of each subtype. CONCLUSIONS: This genetic classification system will help surgeon diagnose patients with FND with unique features and draw a roadmap for their treatment with a better surgical perspective.

Genetically induced redox stress occurs in a yeast model for Roberts syndrome.

Roberts syndrome (RBS) is a multispectrum developmental disorder characterized by severe limb, craniofacial, and organ abnormalities and often intellectual disabilities. The genetic basis of RBS is rooted in loss-of-function mutations in the essential N-acetyltransferase ESCO2 which is conserved from yeast (Eco1/Ctf7) to humans. ESCO2/Eco1 regulate many cellular processes that impact chromatin structure, chromosome transmission, gene expression, and repair of the genome. The etiology of RBS remains contentious with current models that include transcriptional dysregulation or mitotic failure. Here, we report evidence that supports an emerging model rooted in defective DNA damage responses. First, the results reveal that redox stress is elevated in both eco1 and cohesion factor Saccharomyces cerevisiae mutant cells. Second, we provide evidence that Eco1 and cohesion factors are required for the repair of oxidative DNA damage such that ECO1 and cohesin gene mutations result in reduced cell viability and hyperactivation of DNA damage checkpoints that occur in response to oxidative stress. Moreover, we show that mutation of ECO1 is solely sufficient to induce endogenous redox stress and sensitizes mutant cells to exogenous genotoxic challenges. Remarkably, antioxidant treatment desensitizes eco1 mutant cells to a range of DNA damaging agents, raising the possibility that modulating the cellular redox state may represent an important avenue of treatment for RBS and tumors that bear ESCO2 mutations.

Genetic architecture of orbital telorism.

The interocular distance, or orbital telorism, is a distinctive craniofacial trait that also serves as a clinically informative measure. While its extremes, hypo- and hypertelorism, have been linked to monogenic disorders and are often syndromic, little is known about the genetic determinants of interocular distance within the general population. We derived orbital telorism measures from cranial magnetic resonance imaging by calculating the distance between the eyeballs' centre of gravity, which showed a good reproducibility with an intraclass correlation coefficient of 0.991 (95% confidence interval 0.985-0.994). Heritability estimates were 76% (standard error = 12%) with a family-based method (N = 364) and 39% (standard error = 2.4%) with a single nucleotide polymorphism-based method (N = 34 130) and were unaffected by adjustment for height (model II) and intracranial volume (model III) or head width (model IV). Genome-wide association studies in 34 130 European individuals identified 56 significantly associated genomic loci (P < 5 × 10-8) across four different models of which 46 were novel for facial morphology, and overall these findings replicated in an independent sample (N = 10 115) with telorism-related horizontal facial distance measures. Genes located nearby these 56 identified genetic loci were 4.9-fold enriched for Mendelian hypotelorism and hypertelorism genes, underlining their biological relevance. This study provides novel insights into the genetic architecture underlying interocular distance in particular, and the face in general, and explores its potential for applications in a clinical setting.

Expanding the phenotype of ASXL3-related syndrome: A comprehensive description of 45 unpublished individuals with inherited and de novo pathogenic variants in ASXL3.

The study aimed at widening the clinical and genetic spectrum of ASXL3-related syndrome, a neurodevelopmental disorder, caused by truncating variants in the ASXL3 gene. In this international collaborative study, we have undertaken a detailed clinical and molecular analysis of 45 previously unpublished individuals with ASXL3-related syndrome, as well as a review of all previously published individuals. We have reviewed the rather limited functional characterization of pathogenic variants in ASXL3 and discuss current understanding of the consequences of the different ASXL3 variants. In this comprehensive analysis of ASXL3-related syndrome, we define its natural history and clinical evolution occurring with age. We report familial ASXL3 pathogenic variants, characterize the phenotype in mildly affected individuals and discuss nonpenetrance. We also discuss the role of missense variants in ASXL3. We delineate a variable but consistent phenotype. The most characteristic features are neurodevelopmental delay with consistently limited speech, significant neuro-behavioral issues, hypotonia, and feeding difficulties. Distinctive features include downslanting palpebral fissures, hypertelorism, tubular nose with a prominent nasal bridge, and low-hanging columella. The presented data will inform clinical management of individuals with ASXL3-related syndrome and improve interpretation of new ASXL3 sequence variants.

Pathogenic variants in CDH11 impair cell adhesion and cause Teebi hypertelorism syndrome.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca2+ binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.

MASP1-related 3MC syndrome in a patient from Turkey.

3MC syndrome is a rare condition manifesting with typical facial appearance, postnatal growth deficiency, skeletal manifestations, and genitourinary tract anomalies. 3MC is caused by biallelic pathogenic variants in MASP1, COLEC11, or COLEC10. Here, we report an affected subject of Kurdish origin from Turkey presenting with facial dysmorphisms, such as, hypertelorism, blepharophimosis, blepharoptosis, highly arched eyebrows, umbilical hernia, and caudal appendage. These features were compatible with 3MC syndrome. Molecular analysis revealed a novel homozygous pathogenic variant, c.310C > T; p.Gln104Ter in the MASP1 gene, resulting in a premature stop codon. Few subjects with 3MC syndrome have been reported in the literature so far. Thus, detailed study of this subject contributes to the evolving clinical and genetic characterization of 3MC syndrome.

Human model of IRX5 mutations reveals key role for this transcription factor in ventricular conduction.

AIMS: Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. METHODS AND RESULTS: Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. CONCLUSION: Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases.

Craniofacial phenotypes associated with Robinow syndrome.

Robinow syndrome is characterized by mesomelic limb shortening, hemivertebrae, and genital hypoplasia. Due to low prevalence and considerable phenotypic variability, it has been challenging to definitively characterize features of Robinow syndrome. While craniofacial abnormalities associated with Robinow syndrome have been broadly described, there is a lack of detailed descriptions of genotype-specific phenotypic craniofacial features. Patients with Robinow syndrome were invited for a multidisciplinary evaluation conducted by specialist physicians at our institution. A focused assessment of the craniofacial manifestations was performed by a single expert examiner using clinical examination and standard photographic images. A total of 13 patients with clinical and molecular diagnoses consistent with either dominant Robinow syndrome (DRS) or recessive Robinow syndrome (RRS) were evaluated. On craniofacial examination, gingival hyperplasia was nearly ubiquitous in all patients. Orbital hypertelorism, a short nose with anteverted and flared nares, a triangular mouth with a long philtrum, cleft palate, macrocephaly, and frontal bossing were not observed in all individuals but affected individuals with both DRS and RRS. Other anomalies were more selective in their distribution in this patient cohort. We present a comprehensive analysis of the craniofacial findings in patients with Robinow Syndrome, describing associated morphological features and correlating phenotypic manifestations to underlying genotype in a manner relevant for early recognition and focused evaluation of these patients.

An ever-changing landscape in Roberts syndrome biology: Implications for macromolecular damage.

Roberts syndrome (RBS) is a rare developmental disorder that can include craniofacial abnormalities, limb malformations, missing digits, intellectual disabilities, stillbirth, and early mortality. The genetic basis for RBS is linked to autosomal recessive loss-of-function mutation of the establishment of cohesion (ESCO) 2 acetyltransferase. ESCO2 is an essential gene that targets the DNA-binding cohesin complex. ESCO2 acetylates alternate subunits of cohesin to orchestrate vital cellular processes that include sister chromatid cohesion, chromosome condensation, transcription, and DNA repair. Although significant advances were made over the last 20 years in our understanding of ESCO2 and cohesin biology, the molecular etiology of RBS remains ambiguous. In this review, we highlight current models of RBS and reflect on data that suggests a novel role for macromolecular damage in the molecular etiology of RBS.

Juberg-Hayward syndrome and Roberts syndrome are allelic, caused by mutations in ESCO2.

OBJECTIVE: Juberg-Hayward syndrome (JHS; MIM 216100) is a rare autosomal recessive malformation syndrome, characterized by cleft lip/palate, microcephaly, ptosis, hypoplasia or aplasia of thumbs, short stature, dislocation of radial head, and fusion of humerus and radius leading to elbow restriction. A homozygous mutation in ESCO2 has recently been reported to cause Juberg-Hayward syndrome. Our objective was to investigate the molecular etiology of Juberg-Hayward syndrome in two affected Lisu tribe brothers. MATERIALS AND METHODS: Two patients, the unaffected parents, and two unaffected siblings were studied. Clinical and radiographic examination, whole exome sequencing, Sanger sequencing, Western blot analysis, and chromosome testing were performed. RESULTS: Two affected brothers had characteristic features of Juberg-Hayward syndrome, except for the absence of microcephaly. The elder brother had bilateral cleft lip and palate, short stature, humeroradial synostosis, and simple partial seizure with secondary generalization. The younger brother had unilateral cleft lip and palate, short stature, and dislocation of radial heads. The homozygous (c.1654C > T; p.Arg552Ter) mutation in ESCO2 was identified in both patients. The other unaffected members of the family were heterozygous for the mutation. The presence of humeroradial synostosis and radial head dislocation in the same family is consistent with both being in the same spectrum of forearm malformations. Chromosome testing of the affected patients showed premature centromere separation. Western blot analysis showed reduced amount of truncated protein. CONCLUSION: Our findings confirm that a homozygous mutation in ESCO2 is the underlying cause of Juberg-Hayward syndrome. Microcephaly does not appear to be a consistent feature of the syndrome.

Bifid nose as the sole manifestation of BNAR syndrome, a FREM1-related condition.

BNAR syndrome (MIM608980) is a very rare condition: nine cases belonging to three unrelated families were reported since its first description in 2002. The distinctive clinical feature is the bifidity of the tip of the nose and its association with anorectal and/or renal anomalies. Its molecular basis consisting of biallelic FREM1 missense or nonsense mutations was elucidated after studying the original Egyptian family and was confirmed in two families originating from Afghanistan and Pakistan. We describe a fourth family originating from Turkey with signs challenging the diagnostic criteria suggested by the description of the three reported families.

Hydrothorax in fetal cases of Opitz G/BBB diagnosis: Extending the phenotype?

We report two fetal cases carrying a de novo MID1 mutation and presenting with severe hydrothorax, suggesting the expansion of the phenotype of Opitz GBBB syndrome.

Sphenoid sinus agenesis and sella turcica hypoplasia: very rare cases of two brothers with Hamamy syndrome.

Hamamy syndrome (HS) is an autosomal recessive syndrome with a genetic origin that is very rarely observed. The syndrome with craniofacial dysmorphisms, including midface prominence, severe telecanthus, sparse lateral eyebrows, protruding ears, fronto-nasal abnormalities, lacrimal-salivary apparatus agenesis, thin upper vermillion border, myopia, mental retardation, sensorineural hearing impairment, congenital heart anomalies with intraventricular conduction delay, hypochromic microcytic anaemia and skeletal abnormalities of the long bones with recurrent fractures. In this paper, we report a case of two brothers diagnosed with HS at the ages of 25 and 18 years, visited out clinic at different times due to dental reasons. In the radiological examinations, it was observed that both brothers have sphenoid sinuses agenesia, and their sella turcica were smaller than normal. HS may be observed very rarely, and it should be kept in mind that, in addition to various symptoms, it may also cause sphenoid sinus agenesis and sella turcica hypoplasia as shown for the first time in this case report.

Roberts syndrome in an Indian patient with humeroradial synostosis, congenital elbow contractures and a novel homozygous splice variant in ESCO2.

Roberts syndrome (also known as Roberts-SC phocomelia syndrome) is an autosomal recessive developmental disorder, characterized by pre- and postnatal growth retardation, limb malformations including bilateral symmetric tetraphocomelia or mesomelia, and craniofacial dysmorphism. Biallelic loss-of-function variants in ESCO2, which codes for establishment of sister chromatid cohesion N-acetyltransferase 2, cause Roberts syndrome. Phenotypic spectrum among patients is broad, challenging clinical diagnosis in mildly affected individuals. Here we report a 3-year-old boy with a mild phenotype of Roberts syndrome with bilateral elbow contractures, humeroradial synostosis, mild lower limb disparity, and facial dysmorphism. Trio whole-exome sequencing identified the novel biallelic splice variant c.1673+1G>A in ESCO2 in the patient. Aberrant ESCO2 pre-mRNA splicing, reduced relative ESCO2 mRNA amount, and characteristic cytogenetic defects, such as premature centromere separation, heterochromatin repulsion, and chromosome breaks, in patient cells strongly supported pathogenicity of the ESCO2 variant affecting one of the highly conserved guanine-thymine dinucleotide of the donor splice site. Our case highlights the difficulty in establishing a clinical diagnosis in individuals with minor clinical features of Roberts syndrome and normal intellectual and social development. However, next-generation sequencing tools allow for molecular diagnosis in cases presenting with mild developmental defects.

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.