Cleidocranial dysplasia and novel RUNX2 variants: dental, craniofacial, and osseous manifestations

Abstract Cleidocranial dysplasia (CCD) is a skeletal disorder affecting cranial sutures, teeth, and clavicles, and is associated with the RUNX2 mutations. Although numerous patients have been described, a direct genotype-phenotype correlation for RUNX2 has been difficult to establish. Further cases must be studied to understand the clinical and genetic spectra of CCD. Objectives To characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members. Methodology Clinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools. Results Three cases were sporadic and two were familial. Exome sequencing successfully detected the heterozygous pathogenic RUNX2 variants in all affected individuals. Three were novel, comprising a frameshift c.739delA (p.(Ser247Valfs*)) in exon 6 (Patient-1), a nonsense c.901C>T (p.(Gln301*)) in exon 7 (Patient-2 and affected mother), and a nonsense c.1081C>T (p.(Gln361*)) in exon 8 (Patient-3). Two previously reported variants were missense: the c.673C>T (p.(Arg225Trp)) (Patient-4) and c.674G>A (p.(Arg225Gln)) (Patient-5) in exon 5 within the Runt homology domain. Patient-1, Patient-2, and Patient-4 with permanent dentition had thirty, nineteen, and twenty unerupted teeth, respectively; whereas Patient-3 and Patient-5, with deciduous dentition, had normally developed teeth. All patients exhibited typical CCD features, but the following uncommon/unreported phenotypes were observed: left fourth ray brachymetatarsia (Patient-1), normal clavicles (Patient-2 and affected mother), phalangeal malformations (Patient-3), and normal primary dentition (Patient-3, Patient-5). Conclusions The study shows that exome sequencing is effective to detect mutation across ethnics. The two p.Arg225 variants confirm that the Runt homology domain is vital for RUNX2 function. Here, we report a new CCD feature, unilateral brachymetatarsia, and three novel truncating variants, expanding the phenotypic and genotypic spectra of RUNX2 , as well as show that the CCD patients can have normal deciduous teeth, but must be monitored for permanent teeth anomalies.

A novel de novo COL1A1 mutation in a Thai boy with osteogenesis imperfecta born to consanguineous parents

Abstract Osteogenesis imperfecta (OI) is genetically heterogeneous. Mutations in COL1A1 and COL1A2 are responsible for at least 90% of the cases, which are transmitted in an autosomal dominant manner or are de novo events. We identified a Thai boy with OI whose parents were first cousins. Because the proband was the product of a consanguineous marriage, we hypothesized that he might be homozygous for a mutation in a known gene causing a recessive form of OI. Using whole exome sequencing (WES), we did not find any pathogenic mutations in any known gene responsible for an autosomal recessive form of OI. Instead, we identified a COL1A1 frameshift mutation, c.1290delG (p.Gly431Valfs*110) in heterozygosis. By Sanger sequencing, the mutation was confirmed in the proband, and not detected in his parents, indicating that it was a de novo mutation. These findings had implication for genetic counseling. In conclusion, we expanded the mutational spectrum of COL1A1 and provided another example of a de novo pathogenic mutation in heterozygosis in a patient born to consanguineous parents.

Nonsense mutations of the CYBB gene in two Thai families with X-linked chronic granulomatous disease.

X-linked chronic granulomatous disease (X-CGD) is an immunodeficiency disorder characterized by defective intracellular killing of microorganisms due to the neutrophils' inability to generate superoxide ions. Although it is always caused by mutations in the CYBB gene, clinical and molecular characteristics vary in different ethnic backgrounds. Two unrelated Thai boys presented with severe persistent pulmonary infections at the age of two months. Their abnormal dihydrorhodamine (DHR) flow cytometry assays supported the diagnosis of X-CGD. Mutation analysis was performed by polymerase chain reaction (PCR) amplification and sequencing of the entire coding regions of CYBB. Mutations identified were confirmed by restriction enzyme analyses. PCR-sequencing of the entire coding regions of CYBB identified nonsense mutations, 271C>T (R91X) in exon 4 and 456T>A (Y152X) in exon 5, in probands of each family. Both of the patients' mothers were found to be carriers. This observation supports that CYBB is the gene responsible for X-CGD across different populations and nonsense mutations are associated with severe phenotypes.

Prenatal exclusion of Pompe disease by electron microscopy.

Pompe disease is a lysosomal storage disorder caused by alpha-glucosidase deficiency. The disease is characterized by accumulation of glycogen in the lysosomes. The accumulation has unique ultrastructural features which enable a prenatal diagnosis by electron microscopy. We describe prenatal electron microscopic testing in a fetus of a mother whose previous child died of Pompe disease. The disease in the affected child was diagnosed by a decrease in alpha-glucosidase activity of his skin fibroblasts. Electron microscopy of the chorionic villus sample and amniocytes revealed normal findings, thus predicting an unaffected fetus. The study was confirmed by the birth of a normal neonate who was still healthy at the age of 12 months. Electron microscopy is useful in the first and second trimesters to exclude Pompe disease prenatally. This test can be use prenatally and provides families with reassurance.

A novel nonsense mutation, E150X, in the SOX9 gene underlying campomelic dysplasia

Campomelic dysplasia (CD) is an autosomal dominant skeletal malformation syndrome with features including bowed lower limbs with pretibial skin dimpling, hypoplastic scapulae and pelvic bones, and 11 pairs of ribs. Mutations in the SOX9 gene have been identified to cause CD. The gene encodes a transcription factor containing a dimerization, a high mobility group, and a C-terminal transactivation (TA) domain. Up to now, 35 SOX9 mutations have been published. In the present study, we describe a Thai girl with clinically and radiologically typical CD. Direct sequencing analysis of the PCR products for the entire coding region of SOX9 revealed that she was heterozygous for a novel 448G > T in exon 2 of SOX9. The DNA change was expected to result in E150X and loss of the entire TA domain. This result further supports that SOX9 is the only gene, discovered to date, responsible for CD across different populations and that the TA domain is important to the function of the normal SOX9.

A germline mutation in a Thai family with familial multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1, caused by the mutation in the MEN1 gene, is an autosomal dominant disorder with over 95% penetrance characterized by hyperparathyroidism, pancreatic endocrine tumor and pituitary tumor. The authors performed a molecular analysis to identify a mutation in a Thai man with MEN1. He was found to be heterozygous for IVS6 + 1G to A. Two of his three children were also found to carry this mutation. The newly available genetic test for patients with MEN1 in Thailand makes it possible to accurately DNA-based diagnose clinically suspected individuals and their presymptomatic members, which has important therapeutic impacts on them.

Prenatal exclusion of Crouzon syndrome by mutation analysis of FGFR2.

Crouzon Syndrome is an autosomal dominant syndromic craniosynostosis characterized by premature closure of cranial sutures, exophthalmos, and midface hypoplasia. It is caused by multiple mutations in the fibroblast growth factor receptor 2 (FGFR2). We describe prenatal genetic testing of FGFR2 in a fetus of a mother whose previous child had Crouzon Syndrome due to an apparently de novo mutation, S351C. Sequence electropherograms of the exon 10 of FGFR2 encompassing the codon 351 revealed only the normal sequence, thus predicting a very high likelihood of an unaffected fetus. The study was confirmed by the birth of a normal neonate. We report the use of molecular genetic testing to exclude Crouzon Syndrome due to FGFR2 mutation prenatally. Prenatal diagnostic testing for a known mutation is a reasonable option for couples at risk for having a child with Crouzon Syndrome due to germline mosaicism. Molecular testing is more accurate and reliable than ultrasonography and provides families with reassurance.

Molecular diagnosis of dysmorphic syndromes and inherited metabolic disorders in Thailand.

While dysmorphic syndromes and inherited metabolic disorders are individually rare, they collectively account for a significant proportion of illnesses, especially in children. They present clinically in a wide variety of ways, involving virtually any organ or tissue of the body making them relatively difficult to diagnose. However, reaching an accurate diagnosis for children with dysmorphic features and suspected inherited metabolic disorders is important to them and their families both for treatment and for the prevention of disease in other family members. It also makes all the accumulated knowledge available about the relevant condition. Molecular techniques have kindled a revolution in the diagnosis of genetic disorders, including dysmorphic syndromes and inherited metabolic disorders. Molecular methods essentially avoid problems of other techniques. This review exemplifies some of the diseases that can be diagnosed by molecular tools available in Thailand and illustrates some of their benefits.

A RET C634R mutation in a Thai female with multiple endocrine neoplasia type 2A.

Multiple endocrine neoplasia type 2A (MEN 2A) is an autosomal dominant disorder characterized by medullary thyroid carcinoma, pheochromocytoma and primary hyperparathyroidism. The first tumor is usually a medullary thyroid carcinoma. MEN 2A is caused by mutations in the RET proto-oncogene. The detection of mutations in the gene has important diagnostic and therapeutic impacts. Genetic testing of at-risk family members allows one to identify individuals carrying the mutant alleles with very high specificity and sensitivity. Subsequently, total thyroidectomy, recommended at 5 years of age, can be performed in a prophylactic attempt. The authors performed a molecular analysis to identify a mutation in a Thai woman with MEN 2A. She was found to be heterozygous for 1900T>C (C634R). The patient had two daughters who were not found to carry the mutation. The newly available genetic test for patients with MEN 2A in Thailand makes possible accurate DNA-based diagnosis of their at-risk family members before development of the disease, which has important therapeutic impacts for them.

Cystinuria: cause of recurrent renal stones in a 4-year-old girl.

This paper presents the case report of a 4-year and 6-month old girl with cystinuria. She clinically presented with recurrent radiopaque renal stones since the age of 3 years. She received 2 subsequent operations of pyelolithotomy combined with ureterolithotomy at the age of 3 years 6 months, and pyelolithotomy alone at the age of 5 years. She was initially diagnosed as having cystinuria by the presence of hexagonal plate crystals in her acidified urine and positive for the urinary cyanide-nitroprusside test. The diagnosis was confirmed by urinary amino acid analysis using quantitative ion-exchange chromatography which revealed increased amounts of cystine and dibasic amino acids of lysine and ornithine. In spite of maintaining a high fluid intake and alkalinizing urine by giving potassium citrate after the first operation, recurrent renal stones were found. Therefore, after the second operation, D-penicillamine was additionally introduced. During the 18-month follow-up, although there were recurrent renal stones, the rate of stone formation was slower. To the authors' knowledge, this is the first case report in Thailand.

Clinical, pathological, and electron microscopic findings in two Thai children with Pompe disease.

The authors report on a Thai boy who first presented at age 7 months and an unrelated Thai girl in her neonatal period with hypotonia, cardiomegaly and hepatomegaly. Their chest roentgenograms showed markedly enlarged hearts, EKGs showed abnormally shortened PR intervals with gigantic QRS complexes, and electron microscopic studies of their skin samples showed glycogen accumulations surrounded by membranes. The boy died at age 22 months and the girl at age 9 months due mainly to cardiorespiratory failure. Autopsy of the girl showed marked accumulation of glycogen in the liver, heart and numerous additional tissues including her brain. The clinical, pathological, and electron microscopic findings of these two children are consistent with the diagnosis of Pompe disease. Pompe disease is an autosomal recessive disorder of glycogen metabolism resulting from deficiencies in activity of the lysosomal acid alpha-glucosidase. Definite diagnosis of the disease can be made from a biochemical test or a mutation analysis. To the authors' knowledge, no service laboratories in Thailand offer the tests. Because Thai children have occasionally been reported to be affected by Pompe disease, an attempt to establish a definite diagnostic test for Pompe disease in Thailand should be encouraged. With a definite diagnosis, the proper genetic counseling and prenatal diagnosis could be offered to the families.