Patra Yeetong

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.

A newly identified locus for benign adult familial myoclonic epilepsy on chromosome 3q26.32-3q28

Benign Adult Familial Myoclonic Epilepsy (BAFME) is an autosomal dominant disorder characterized by adult-onset cortical tremor or action myoclonus predominantly in the upper limbs, and generalized seizures. We investigated a Thai BAFME family. Clinical and electrophysiological studies revealed that 13 were affected with BAFME. There were a total of 24 individuals studied. Genetic analysis by genome-wide linkage study (GWLS) was performed using 400 microsatellite markers and excluded linkage of the previous BAFME loci, 8q23.3-q24.1, and 2p11.1-q12.2. GWLS showed that the disease-associated region in our Thai family was linked to a newly identified locus on chromosome 3q26.32-3q28. This locus represents the fourth chromosomal region for BAFME.

Identification of mutations in the SRD5A2 gene in Thai patients with male pseudohermaphroditism.

OBJECTIVE To describe two unrelated Thai patients with suspected 5alpha-reductase type 2 deficiency and perform mutation analysis of the SRD5A2 gene. DESIGN Case report. SETTING A pediatric endocrinology clinic at a university hospital. PATIENT(S) Two unrelated patients with 46,XY karyotype, born with ambiguous genitalia, were studied. One was reared as a boy and the other was reared as a girl. INTERVENTION(S) The entire coding regions of the SRD5A2 gene were assessed by polymerase chain reaction (PCR) and sequencing analysis. MAIN OUTCOME MEASURE(S) Molecular characterization of the SRD5A2 gene. RESULT(S) Four different pathogenic mutations (three missense and one nonsense) were identified. These were located at exon 1 (p.Q6X and p.L20P), exon 3 (p.G183S), and exon 4 (p.G203S). The T>C transition (c.59T>C) resulting in a leucine-to-proline substitution at codon 20 (p.L20P) has not been previously described and was not detected in 100 unaffected, ethnic-matched control chromosomes. In addition, p.G183S, previously identified only among patients from mixed African-European ancestry and in the Dominican Republic, was also detected in a Thai patient. CONCLUSION(S) This study demonstrates that the SRD5A2 gene is responsible for 5alpha-reductase type 2 deficiency across different populations and emphasizes the important role of genetic testing for the definite diagnosis and genetic counseling before gender assignment or any surgical intervention.

Three novel mutations of the IRF6 gene with one associated with an unusual feature in Van der Woude syndrome.

Van der Woude syndrome (VWS) is a dominantly inherited disorder characterized by cleft lip with or without cleft palate and lip pits. It remains the most common syndromic form of oral clefts. Mutations in the interferon regulatory factor 6 (IRF6) gene have been identified in patients with VWS. We reported three unrelated families with lower lip anomalies. Two had lower lip pits, a cardinal sign of VWS, but the other had a heart‐shaped mass on lower lip without pits, oral clefts, or hypodontia. This isolated anomaly has not been previously observed in VWS. We performed mutation analysis by PCR‐sequencing the entire coding region of the IRF6 gene. Three potentially pathogenic mutations, c.145C>T (p.Q49X), c.171T>G (p.F57L), and 1306C>G (p.L436V) were successfully identified. All the missense mutations were not detected in 100 unaffected ethnic‐matched control chromosomes and have never been previously reported. The p.Q49X and p.F57L mutations were located in the highly conserved DNA binding domain while the p.L436V was located at the carboxy‐terminal region. This study reported an undescribed clinical feature of VWS and three novel mutations, expanding the phenotypic spectrum of VWS and mutational spectrum of IRF6.

Two novel mutations including a large deletion of the SLC4A11 gene causing autosomal recessive hereditary endothelial dystrophy

Congenital hereditary endothelial dystrophy (CHED) is an inherited disorder of the corneal endothelium characterised by bilateral non-inflammatory corneal clouding ranging from a diffuse haze to a ground-glass appearance. CHED can be inherited in an autosomal dominant (CHED1) or recessive (CHED2) manner. CHED2 usually presents at birth or early infancy. Bilateral corneal clouding can lead to visual impairment often accompanied by nystagmus in CHED2 patients requiring corneal transplantation.1 Mutations in the solute carrier family 4 member 11 ( SLC4A11 ) gene have been identified in most patients with CHED2. With PCR sequencing of the entire coding and putative promoter regions of SLC4A11 , there were, however, some clinically confirmed CHED2 patients with undetected SLC4A11 mutations.2 Three affected siblings with CHED2 from a non-consanguineous Thai family were seen at the age of 7, 17 and 20 years, respectively. A diagnosis of CHED2 was made by clinical features, histopathological and confocal microscopic findings. All had corneal haze since birth. Nystagmus was present in the 20-year-old brother and the 7-year-old sister. None had sensorineural hearing loss. Both parents had clear corneas and denied a family history of corneal disorders. To identify the genetic defects, we first performed PCR sequencing covering the entire coding region of SLC4A11 . A novel c.778A>G mutation resulting …

Two novel CTNS mutations in cystinosis patients in Thailand.

Cystinosis is an autosomal recessive disorder characterized by defective transport of cystine across the lysosomal membrane and resulting in renal, ophthalmic, and other organ abnormalities. Mutations in the CTNS gene cause a deficiency of the transport protein, cystinosin. We performed mutation analysis of CTNS in six cystinosis patients from four families in Thailand. Using PCR sequencing of the entire coding regions, we identified all eight mutant alleles, including two mutations, p.G309D and p.Q284X, that have not been previously reported. This study expands the mutational and population spectrum of nephropathic cystinosis.

Primary hyperoxaluria type 1 and brachydactyly mental retardation syndrome caused by a novel mutation in AGXT and a terminal deletion of chromosome 2

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder caused by mutations in the alanine:glyoxylate aminotransferase (AGXT) gene, located on chromosome 2q37. Mutant AGXT leads to excess production and excretion of oxalate, resulting in accumulation of calcium oxalate in the kidney, and progressive loss of renal function. Brachydactyly mental retardation syndrome (BDMR) is an autosomal dominant disorder, caused by haploinsufficiency of histone deacetylase 4 (HDAC4), also on chromosome 2q37. It is characterized by skeletal abnormalities and developmental delay. Here, we report on a girl who had phenotypes of both PH1 and BDMR. PCR‐sequencing of the coding regions of AGXT showed a novel missense mutation, c.32C>G (p.Pro11Arg) inherited from her mother. Functional analyses demonstrated that it reduced the enzymatic activity to 31% of the wild‐type and redirected some percentage of the enzyme away from the peroxisome. Microsatellite and array‐CGH analyses indicated that the proband had a paternal de novo telomeric deletion of chromosome 2q, which included HDAC4. To our knowledge, this is the first report of PH1 and BDMR, with a novel AGXT mutation and a de novo telomeric deletion of chromosome 2q.

A Frameshift Mutation in PEN-2 Causes Familial Comedones Syndrome

Background: Familial comedones without dyskeratosis are a rare autosomal dominant skin disorder, characterized by the occurrence of comedones that are distributed all over the body with specific features. We have previously reported two Thai families with familial comedones with expanded phenotypic spectrum. However, its genetic defect and pathogenesis remain unknown. Objective: To explore the molecular defect causing familial comedones. Methods: Whole-genome linkage analysis and whole-exome sequencing in family I were performed. Results: We identified a heterozygous one-base pair insertion, c.84_85insT (p. L28FfsX93) in PEN-2, located within the linked region on chromosome 19. PCR-Sanger sequencing confirmed the identified mutation. The mutation segregated with the disease phenotype in family I and was fully penetrant. This similar mutation was also present in the unrelated affected individual from family II. Quantitative PCR revealed increased mRNA expression of PEN-2 in leukocytes of affected individuals. Conclusion: We for the first time identify PEN-2 as the causative gene of familial comedones.

Adaptive immune defects in a patient with leukocyte adhesion deficiency type III with a novel mutation in FERMT3

To the Editor, Leukocyte adhesion deficiency (LAD) is a rare primary immunodeficiency disease characterized by impairment of phagocyte adhesion (1–3). Three subtypes have been classified by distinct phases of the adhesion cascade. LAD-III is caused by defects in signaling pathways used for integrin activation in all hematopoietic cell types leading to recurrent infections with poor platelet aggregation resembling Glanzmann’s thrombasthenia (4). Mutations in FERMT3 have been identified to underlie LAD-III (4, 5). FERMT3 encodes kindlin-3, one of the focal adhesion proteins which contain a FERM domain located at the carboxyl terminus binding to b-integrin cytoplasmic tails. This molecule cooperates with the cytoskeletal protein talin leading to integrin activation. It also stabilizes active conformations of the integrin subunits and the ligand binding (5, 6). Evidently, integrins are widely expressed in many cell types including T and B lymphocytes. Defects in integrin function therefore could lead to both innate and adaptive immune dysfunctions. However, almost all reported cases of LAD-III only had innate immune defects. Here, we describe a female Thai patient who was diagnosed with LAD-III, yet presenting with a mild atypical phenotype in which a humoral immune defect was detected. Our patient was the second child of consanguineous parents who were first cousins. The pedigree of the family is shown in Fig. 1a. She presented with early-onset severe gram-negative infections, thrombasthenia, hepatosplenomegaly, and defective wound healing. Between three and 8 months old, she experienced four episodes of bacterial pneumonia with sepsis. Firstly, she had severe pneumonia and subsequently developed acute respiratory distress syndrome. Cultures of tracheal suction specimens revealed Acinetobacter baumannii. Salmonella spp. was also reported from stool samples when she was found to have diarrhea. In the second episode of pneumonia, A. baumannii was reported again from specimens obtained by tracheal suctioning. Pseudomonas aeruginosa was isolated from ear discharge. Thirdly, the patient had pneumonia with septic shock. Tracheal suction cultures revealed Streptococcus mitis and Escherichia coli. Finally, necrotizing pneumonia was reported. The patient’s blood culture was positive for P. aeruginosa. She had the ability to form pus, although minimal, and umbilical cord separation occurred at the age of 9 days. After prolonged courses of antibiotics, the patient had developed mucocutaneous candidiasis. She did not suffer from invasive fungal infections, as described in other patients with LAD-III (3, 7). Her bleeding symptoms were mild and appeared only during episodes of infections, while spontaneous intracranial bleeding and/or massive pulmonary hemorrhage have been reported in patients with typical LAD-III (3, 7). Initial investigations and immunologic assessment at the age of 5 months revealed persistent leukocytosis with neutrophilia, anemia, and thrombocytopenia. A complete blood count showed a hematocrit of 28% (29–42), white blood cell count of 45,430 cells/mm (6000–17,500), neutrophils of 25,440 cells/ mm (4000–12,000), lymphocytes of 10,903 cells/mm (2000– 17,000), and platelets of 109,000 cells/mm (300,000–700,000). Flow cytometric analysis of lymphocyte populations demonstrated normal numbers of total T cells (CD3+), CD4+ T cells, CD8+ T cells, B cells (CD19+), and NK cells (CD16+56+).

Splicing analysis of CYP11B1 mutation in a family affected with 11β-hydroxylase deficiency: case report

BackgroundCongenital adrenal hyperplasia (CAH) due to steroid 11β-hydroxylase deficiency (11β-OHD) is a rare form of CAH associated with low renin hypertension, hypokalemia, hyperandrogenemia and ambiguous genitalia in affected females. Herein we describe the clinical, hormonal and molecular characteristics of two Uzbekistan siblings with 11β-OHD and analyze the effects of a splicing mutation.Case presentationA 46,XX girl presented with genital ambiguity and low renin hypertension; her 46,XY brother presented with precocious puberty. Hormonal studies suggested 11β-OHD. Mutation analysis was performed by PCR followed by Sanger sequencing of the entire coding regions and their flanking introns of the CYP11B1 gene. Mutation analysis showed that both patients were compound heterozygous for IVS7 + 1G > A, and c.421C > T. Although the identified mutations have been previously described, this is, to our knowledge, the first report of these mutations in compound heterozygotes. A minigene assay was used to determine the effects of the splicing mutation. The constructs containing either the wild-type or the splice-site mutant CYP11B1 genomic DNA of exons-introns 6–9 were transfected into COS-7 cells; subsequently, RNA splicing was assessed by reversed transcribed-PCR of CYP11B1 complementary DNA. The minigene assay revealed that the IVS7 + 1G > A mutation resulted in two shorter incorrectly spliced products; one skipping the exon 7 and the other skipping the exons 7–8. The c.421C > T mutation leads to the introduction of a premature stop codon at residue 141 (p.R141X). These mutations are expected to code non-functional proteins.ConclusionCompound heterozygous mutations (IVS7 + 1G > A and p.R141X) in the CYP11B1 gene were found to cause 11β-OHD. The IVS7 + 1G > A mutation causes aberrant splicing of CYP11B1 leading to exon skipping. This finding could facilitate the future novel therapies targeted on splicing modulation to treat human disease.