Assessment of a novel variation in DHODH gene causing Miller syndrome: The first report in Chinese population.

BACKGROUND: Miller syndrome is a rare type of postaxial acrofacial dysostosis caused by biallelic mutations in the DHODH gene, which is characterized mainly by craniofacial malformations of micrognathia, orofacial clefts, cup-shaped ears, and malar hypoplasia, combined with postaxial limb deformities like the absence of fifth digits. METHODS: In this study, a prenatal case with multiple orofacial-limb abnormities was enrolled, and a thorough clinical and imaging examination was performed. Subsequently, genetic detection with karyotyping, chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) was carried out. In vitro splicing analysis was also conducted to clarify the impact of one novel variant. RESULTS: The affected fetus displayed typical manifestations of Miller syndrome, and WES identified a diagnostic compound heterozygous variation in DHODH, consisting of two variants: exon(1-3)del and c.819 + 5G > A. We conducted a further in vitro validation with minigene system, and the result indicated that the c.819 + 5G > A variant would lead to an exon skipping in mRNA splicing. CONCLUSIONS: These findings provided with the first exonic deletion and first splice site variant in DHODH, which expanded the mutation spectrum of Miller syndrome and offered reliable evidence for genetic counseling to the affected family.

Dihydroorotate dehydrogenase mutations lead to changes of protein and function in Miller syndrome / 重庆医学

Objective To observe the changes of corresponding proteins and function based on known clinical dihydroorotate dehydrogenase(DHODH) mutation types,i.e.,G202A,R346W and R135C in the patients with Miller syndrome.Methods HeLacell lines stably expressing Miller syndrome pathogenic mutation types G202A,R346W and R135C were established.Then the mitochondrial localization function,protein stability and enzyme activity of corresponding proteins were studied by the immunohistochemistry and mitochondrial layered positioning.Results The mitochondrial localization function of 3 kinds of DHODH mutation were not affected,which existed in the mitochondrial inner membrane after expression.The mutant G202A and R346W protein stability was reduced;the mutant R135C protein was stable,but base induced enzyme activity injury caused the deficiency of corresponding enzymatic activity.Conclusion The DHODH function injury may be related with the symptoms in Miller syndrome.

Study on effects of DHODH deficiency on mitochondrial function and differentiation and maturation in osteoblast cells / 重庆医学

Objective To observe the changes of the skeletal development related cells after dihydroorotate dehydrogenase (DHODH) deficiency.Methods The DHODH expression in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was inhibited by specific small interfering RNAs (siRNAs),and cell proliferation,ATP production and expression levels of bone-related genes were investigated in these cells.Results After reducing the DHODH expression by using specific siRNAs,cell proliferation was inhibited and cell cycle was arrested at G1/S stage.In addition,the ATP production was reduced in whole cells,especially in mitochondria.Furthermore,the expression levels of Runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs in the DHODH inhibition group were decreased compared with the control group.Conclusion Inhibiting DHODH protein affects the differentiation and maturation of osteoblasts.The mitochondrial dysfunction in osteoblasts may be one of causes leading to the abnormal bone formation in Miller syndrome.

Elevated plasma dihydroorotate in Miller syndrome: Biochemical, diagnostic and clinical implications, and treatment with uridine.

BACKGROUND: Miller syndrome (post-axial acrofacial dysostosis) arises from gene mutations for the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). Nonetheless, despite demonstrated loss of enzyme activity dihydroorotate (DHO) has not been shown to accumulate, but paradoxically urine orotate has been reported to be raised, confusing the metabolic diagnosis. METHODS: We analysed plasma and urine from a 4-year-old male Miller syndrome patient. DHODH mutations were determined by PCR and Sanger sequencing. Analysis of DHO and orotic acid (OA) in urine, plasma and blood-spot cards was performed using liquid chromatography-tandem mass spectrometry. In vitro stability of DHO in distilled water and control urine was assessed for up to 60h. The patient received a 3-month trial of oral uridine for behavioural problems. RESULTS: The patient had early liver complications that are atypical of Miller syndrome. DHODH genotyping demonstrated compound-heterozygosity for frameshift and missense mutations. DHO was grossly raised in urine and plasma, and was detectable in dried spots of blood and plasma. OA was raised in urine but undetectable in plasma. DHO did not spontaneously degrade to OA. Uridine therapy did not appear to resolve behavioural problems during treatment, but it lowered plasma DHO. CONCLUSION: This case with grossly raised plasma DHO represents the first biochemical confirmation of functional DHODH deficiency. DHO was also easily detectable in dried plasma and blood spots. We concluded that DHO oxidation to OA must occur enzymatically during renal secretion. This case resolved the biochemical conundrum in previous reports of Miller syndrome patients, and opened the possibility of rapid biochemical screening.

Dihydroorotate dehydrogenase depletion hampers mitochondrial function and osteogenic differentiation in osteoblasts.

Mutation of the dihydroorotate dehydrogenase (DHODH) gene is responsible for Miller syndrome, which is characterized by craniofacial malformations with limb abnormalities. We previously demonstrated that DHODH was involved in forming a mitochondrial supercomplex and that mutated DHODH led to protein instability, loss of enzyme activity, and increased levels of reactive oxygen species in HeLa cells. To explore the etiology of Miller syndrome in more detail, we investigated the effects of DHODH inhibition in the cells involved in skeletal structure. Dihydroorotate dehydrogenase in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was knocked down by specific small interfering RNAs (siRNAs), and cell proliferation, ATP production, and expression of bone-related genes were investigated in these cells. After depletion of DHODH using specific siRNAs, inhibition of cell proliferation and cell cycle arrest occurred in MC3T3-E1 cells. In addition, ATP production was reduced in whole cells, especially in mitochondria. Furthermore, the levels of runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs were lower in DHODH siRNA-treated cells compared with controls. These data suggest that depletion of DHODH affects the differentiation and maturation of osteoblasts. This study shows that mitochondrial dysfunction by DHODH depletion in osteoblasts can be directly linked to the abnormal bone formation in Miller syndrome.

Orotic Acid, More Than Just an Intermediate of Pyrimidine de novo Synthesis.

It is timely to consider the many facets of the small molecule orotic acid (OA), which is well-known as an essential intermediate of pyrimidine de novo synthesis. In addition, it can be taken up by erythrocytes and hepatocytes for conversion into uridine and for use in the pyrimidine recycling pathway. We discuss the link between dietary orotate and fatty liver in rats, and the potential for the alleviation of neonatal hyperbilirubinaemia. We address the development of orotate derivatives for application as anti-pyrimidine drugs, and of complexes with metal ions and organic cations to assist therapies of metabolic syndromes. Recent genetic data link human Miller syndrome to defects in the dihydroorotate dehydrogenase (DHODH) gene, hence to depleted orotate production. Another defect in pyrimidine biosynthesis, the orotic aciduria arising in humans and cattle with a deficiency of UMP synthase (UMPS), has different symptoms. More recent work leads us to conclude that OA may have a role in regulating gene transcription.

Orodental findings in postaxial acrofacial dysostosis.

We report a new case of postaxial acrofacial dysostosis (Miller) syndrome with expanded profile. The patient presented with unusual orofacial and digital anomalies along with mental retardation. This report emphasizes the recognized features of the syndrome as well as describes intraoral findings that could aid in the diagnosis and management of these patients.

Facial dysostoses: Etiology, pathogenesis and management.

Approximately 1% of all live births exhibit a minor or major congenital anomaly. Of these approximately one-third display craniofacial abnormalities which are a significant cause of infant mortality and dramatically affect national health care budgets. To date, more than 700 distinct craniofacial syndromes have been described and in this review, we discuss the etiology, pathogenesis and management of facial dysostoses with a particular emphasis on Treacher Collins, Nager and Miller syndromes. As we continue to develop and improve medical and surgical care for the management of individual conditions, it is essential at the same time to better characterize their etiology and pathogenesis. Here we describe recent advances in our understanding of the development of facial dysostosis with a view towards early in utero identification and intervention which could minimize the manifestation of anomalies prior to birth. The ultimate management for any craniofacial anomaly however, would be prevention and we discuss this possibility in relation to facial dysostosis.

Síndrome 3-M: descripción clínica-radiológica: caso clínico / 3-M syndrome: clinical and radiological description. Case report

Introducción: El síndrome 3M combina retardo de crecimiento prenatal y postnatal severo, dismorfias faciales (semeja facies "melancólica") y anomalías radiológicas. Es una enfermedad infrecuente de la que hasta el momento se han descrito alrededor de 200 casos. "3M" se refiere a las iniciales de los tres autores que describieron este síndrome. Los rasgos faciales característicos son: cabeza relativamente grande, dolicocefalia, abombamiento frontal, cara triangular, mentón prominente, nariz antevertida, labios gruesos, cejas gruesas, surco nasolabial largo e hipoplasia de tercio medio de cara. Los hallazgos radiológicos, que van apareciendo con la edad son costillas y huesos largos finos y delgados, y cuerpos vertebrales altos. El síndrome 3M se transmite como un rasgo autosómico recesivo y es genéticamente heterogéneo. Objetivo: Descripción del caso clínico de una nina actualmente de 10 años de edad en el que se confirmó este síndrome. Caso clínico: Nina referida a Genética a los 15 meses de vida, por talla baja severa, dismorfias y malformaciones. Su seguimiento clínico y radiológico permitió plantear y confirmar este diagnóstico. Conclusión: En ocasiones sólo el seguimiento longitudinal de pacientes con talla baja severa permite que se evidencien alteraciones sugerentes de un diagnóstico específico. La certificación del diagnóstico favorece un adecuado manejo clínico y consejería genética a los padres.

Introduction: 3M syndrome combines severe prenatal and postnatal growth delay, facial dysmorphism (resembles melancholy facies) and radiological abnormalities. It is a rare disease with 200 cases reported so far. "3M" refers to the initials of the three authors who first described this syndrome. The characteristic facial features are: relatively large head, dolichocephaly, frontal bossing, a triangular face, pointed chin, upturned nose, full lips, full eyebrows, long philtrum and hypoplastic midface. radiological findings which appear with age, include slender long bones and ribs and tall vertebral bodies. 3M syndrome is transmitted as an autosomal recessive trait and is genetically heterogeneous. Objective: Report the clinical case of a girl, now 10 years of age, diagnosed with the syndrome. Case report: An infant girl, 15 months old, was referred to Genetics Clinic due to severe short stature, dysmorphic features and malformations. Her clinical and radiological follow-up led to propose and confirm this diagnosis. Conclusion: Sometimes only longitudinal monitoring of patients with severe short stature evidence abnormalities suggesting a specific diagnosis. The right diagnosis results in suitable clinical care and genetic counseling to parents.