Han-Wook Yoo

PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype–phenotype correlation in Korean patients with Noonan syndrome

AbstractAfter 2006, germline mutations in the KRAS, SOS1, and RAF1 genes were reported to cause Noonan syndrome (NS), in addition to the PTPN11 gene, and now we can find the etiology of disease in approximately 60–70% of NS cases. The aim of this study was to assess the correlation between phenotype and genotype by molecular analysis of the PTPN11, SOS1, KRAS, and RAF1 genes in 59 Korean patients with NS. We found disease-causing mutations in 30 (50.8%) patients, which were located in the PTPN11 (27.1%), SOS1 (16.9%), KRAS (1.7%), and RAF1 (5.1%) genes. Three novel mutations (T59A in PTPN11, K170E in SOS1, S259T in RAF1) were identified. The patients with PTPN11 mutations showed higher prevalences of patent ductus arteriosus and thrombocytopenia. The patients with SOS1 mutations had a lower prevalence of delayed psychomotor development. All patients with RAF1 mutations had hypertrophic cardiomyopathy. Typical facial features and auxological parameters were, on statistical analysis, not significantly different between the groups. The molecular defects of NS are genetically heterogeneous and involve several genes other than PTPN11 related to the RAS-MAPK pathway.

Spectrum of Mutations in Noonan Syndrome and Their Correlation with Phenotypes

OBJECTIVES To investigate mutation spectrums and their correlations to phenotypes in Noonan syndrome (NS) and NS-related disorders that share functional alterations of the Ras-mitogen-activated protein kinase pathway. STUDY DESIGN Clinical characteristics and genotypes of 10 previously known and 2 candidate genes, SPRY1-4 and SPRED1, were investigated in 59 patients with NS, 17 with cardiofaciocutaneous syndrome, 5 with Costello syndrome, and 2 with LEOPARD syndrome. RESULTS PTPN11 (39.0%), SOS1 (20.3%), RAF1 (6.8%), KRAS (5.1%), and BRAF (1.7%) mutations were identified in NS; BRAF (41.2%), SHOC2 (23.5%), and MEK1 (5.9%) mutations in cardiofaciocutaneous syndrome; and HRAS and PTPN11 mutations in Costello syndrome and LEOPARD syndrome, respectively. No additional mutations were identified in 28.9% of NS and 35.3% of cardiofaciocutaneous syndrome. Functional characterizations of 2 RAF1 novel variants, p.P261T and p.S259T, and one SOS1 variant, p.K170E, showed enhanced activity of Ras-mitogen-activated protein kinase pathway. Normal stature was frequent in SOS1 mutations, hypertrophic cardiomyopathy in RAF1, and developmental delay in RAF1, BRAF, or SHOC2 mutations. CONCLUSIONS By identifying genotype-phenotype correlations, our study highlights the role of molecular genetic testing in the process of differential diagnosis of NS and NS-related disorders. Pathophysiologies that underlie these correlations are needed to be investigated in terms of their effects on Ras-mitogen-activated protein kinase pathway.

Clinical and Functional Characteristics of a Novel Heterozygous Mutation of the IGF1R Gene and IGF1R Haploinsufficiency due to Terminal 15q26.2->qter Deletion in Patients with Intrauterine Growth Retardation and Postnatal Catch-Up Growth Failure

CONTEXT Mutations in the IGF1R gene result in intrauterine growth retardation and postnatal growth failure. OBJECTIVE The objective of this study was to describe the clinical features of subjects with a mutation in the IGF1R gene and to evaluate the molecular and functional characteristics of a novel IGF1R mutation. SUBJECTS Three children with unexplained intrauterine growth retardation (birth weight <-1.5 SD score) and persistent short stature (<-2.0 SD score) were included in the study. METHODS Auxological and endocrinological profiles were measured. All coding regions, including the intron-exon boundaries of the IGF1R gene, were amplified via PCR and directly sequenced. To study the functional effect of the IGF1R gene mutation on IGF-I signaling, total IGF1R protein expression, and IGF-I-dependent Akt and ERK phosphorylation were assessed by Western blotting. RESULTS Two children and their father possessed a novel c.420del (p.A110fsX20) mutation in exon 2 of the IGF1R gene. After recombinant human GH therapy, the growth deficit decreased in these two children. Our data show that IGF-I-induced autophosphorylation of the phosphorylated tyrosine and phosphorylated Akt of IGF1R increased in a dose-dependent manner but did so less efficiently in patients. Array comparative genomic hybridization of chromosome 15 identified a heterozygous deletion of 15q26.2 to 15qter in subject 3. CONCLUSIONS The novel heterozygous mutation described in this study reduced IGF1R expression and represents haploinsufficiency of the IGF1R gene. Our results indicate that this mutation in the IGF1R gene leads to abnormalities in the function of IGF1R and also retards intrauterine and subsequent growth in humans.

High incidence of PRSS1 and SPINK1 mutations in Korean children with acute recurrent and chronic pancreatitis.

Objectives: We evaluated the frequencies and clinical consequences of mutations in the genes encoding cationic trypsinogen, serine protease 1 (PRSS1), and serine protease inhibitor Kazal type 1 (SPINK1) in children with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). Patients and Methods: The study population consisted of 32 children with ARP or CP and 28 healthy controls. We analyzed clinical data and the sequences of the entire coding region and the intron-exon boundaries of the PRSS1 and SPINK1 genes from each patient. Results: Fifteen (46.9%) of the 32 patients had at least 1 PRSS1 or SPINK1 mutation. Four (12.5%) of the 32 patients carried the p.N29I, p.R122H, or p.N29T mutation or a p.G208A variant of the PRSS1 gene in a heterozygote state. Eleven (34.4%) of the 32 patients carried either the IVS3 + 2T>C or p.N34S mutation of the SPINK1 gene. No PRSS1 or SPINK1 mutations were identified in the control group. In particular, mutations were identified in 4 of our patients who experienced pancreas divisum with CP, whereas the remaining 2 patients with pancreas divisum and ARP did not have mutation. Conclusions: The frequencies of the PRSS1 and SPINK1 mutations are relatively high in Korean children with ARP or CP. Mutations in the PRSS1 and SPINK1 genes are highly associated with the development of childhood ARP or CP. Our findings suggest that patients with genetic mutations combined with pancreas divisum tend to develop CP early.

Endocrine Manifestations of Chromosome 22q11.2 Microdeletion Syndrome

Background: Endocrine abnormalities, including hypocalcemia, thyroid dysfunction, and short stature, are associated with chromosome 22q11.2 microdeletion syndrome. This study was undertaken to examine the frequencies and clinical features of endocrine abnormalities in patients with 22q11.2 microdeletion syndrome. Methods: We analyzed 61 patients with 22q11.2 microdeletion syndrome diagnosed based on the verification of microdeletion by fluorescent in situ hybridization (FISH) using a probe of the DiGeorge syndrome critical region (TUPLE1) at 22q11.2 and a control probe, ARSA at 22q13. Serum total calcium, phosphorus, and intact parathyroid hormone (PTH) levels were measured, thyroid function test was performed, and serum IGF-1 and IGFBP-3 levels were also estimated. Height and weight of patients were compared with individual chronological ages. Results: Hypocalcemia was found in 20 patients (32.8%), and overt hypoparathyroidism in 8 (13.1%). Two patients (3.3%) showed autoimmune thyroid diseases, 1 each with Graves’ disease and Hashimoto thyroiditis. Ten patients (16.4%) were below the third percentile in height, but the serum IGF-1 level was normal in 9 out of these 10 patients. Conclusion: Our findings show that patients with chromosome 22q11.2 microdeletion syndrome present with variable endocrine manifestations and variable clinical phenotypes. In addition to FISH analysis, careful endocrine evaluations are required in patients with this microdeletion syndrome, particularly for those with hypoparathyroidism or thyroid dysfunction.

Mutations of the GLA gene in Korean patients with Fabry disease and frequency of the E66Q allele as a functional variant in Korean newborns

Fabry disease is caused by an α-galactosidase A (GLA) deficiency. In this study, we identified 28 unrelated Korean families with Fabry disease with 25 distinct mutations in the GLA gene including six novel mutations (p.W47X, p.C90X, p.D61EfsX32, IVS4−11T>A, p.D322E and p.W349). Notably, five subjects from four unrelated families carried the p.E66Q variant, previously known as a pathogenic mutation in atypical Fabry disease. Among these patients, only one had proteinuria and two had hypertrophic cardiomyopathy without any other systemic manifestation of Fabry disease. Substantial residual GLA activity was shown both in the leukocytes of p.E66Q patients (19.0–30.3% of normal activity) and in transiently overexpressed COS-7 cells (43.8±3.03% of normal activity). Although GLA harboring p.E66Q is unstable at neutral pH, the enzyme is efficiently expressed in the lysosomes of COS-7 cells. The location of p.E66 is distant from both the active site and the dimer interface, and has a more accessible surface area than have other mutations of atypical Fabry disease. In addition, the allele frequency of p.E66Q determined in 833 unrelated Korean individuals was remarkably high at 1.046% (95% confidence interval, 0.458–1.634%). These results indicate that p.E66Q is a functional polymorphism rather than a pathogenic mutation.

Clinical Characteristics and Molecular Analysis of PIT1, PROP1,LHX3, and HESX1 in Combined Pituitary Hormone Deficiency Patients with Abnormal Pituitary MR Imaging

Background/Aims: Many genes encoding pituitary transcription factors involved in the formation of the pituitary gland are identified. Different mutations in these genes have been reported in patients with familial combined pituitary hormone deficiency (CPHD). This study was undertaken to analyze PIT1, PROP1, LHX3, and HESX1 in 12 CPHD patients with abnormal pituitary magnetic resonance imaging (MRI). Since embryonic development of the pituitary requires the coordinated expression of specific transcription factors, we postulated the presence of mutations in PIT1, PROP1, LHX3, and HESX1 genes. Methods: Anterior pituitary function was evaluated. Each gene was PCR amplified exon by exon, and subsequently sequenced. Results: In all cases, MRI examination showed abnormal pituitary gland development featuring ectopic neurohypophysis, hypoplastic anterior lobe, empty sella, and septo-optic dysplasia. Endocrinologically, all patients revealed multiple pituitary hormone deficiency including growth hormone, thyroid stimulating hormone, luteinizing hormone, follicular stimulating hormone and adrenocorticotropin. They were all sporadic cases without a positive family history. None of disease-causing specific mutations were identified in PIT1, PROP1, LHX3, and HESX1 genes of 12 sporadic CPHD patients with abnormal pituitary imaging. However, 2 novel polymorphisms were found in PROP1 gene: IVS1+3 A→G and 27 T→C (Ala9Ala) in exon 1. Their allele frequencies in patients and normal controls were not statistically different. Overall, allele frequencies of these polymorphisms were as follows: for the IVS1+3 A→G polymorphism, the allele frequency of A was 54%, and 46% for G, with 58% of an A/G heterozygosity. For the 27 T→C (Ala9Ala) polymorphism, the allele frequency of T was 46%, and 54% for G, with 42% of a T/C heterozygosity. Conclusions: Mutations of PIT1, PROP1, LHX3, and HESX1 genes are very rare in sporadic CPHD patients with abnormal pituitary MRI.

Identification of a novel mutation in the GLUT2 gene in a patient with Fanconi-Bickel syndrome presenting with neonatal diabetes mellitus and galactosaemia

We describe a patient with Fanconi-Bickel syndrome diagnosed by clinical manifestations and the identification of a novel mutation in the GLUT 2 gene. She was initially diagnosed with neonatal diabetes mellitus due to hyperglycaemia and glycosuria at 3 days of life. In addition, newborn screening for galactosaemia revealed hypergalactosaemia. Thereafter, she was managed with lactose-free milk and insulin therapy. However, she failed to grow and her liver became progressively enlarged. Her liver function deteriorated with increased prothrombin time. A liver biopsy done at age 9 months showed micronodular cirrhosis with marked fatty changes and she succumbed to hepatic failure with pneumonia at 10 months of age. DNA sequencing analysis of the GLUT 2 gene using her genomic DNA revealed a novel mutation in codon 5, lysine5 stop(K5X).

The IL-10 (-627 A/C) promoter polymorphism may be associated with coronary aneurysms and low serum albumin in korean children with kawasaki disease

Kawasaki disease (KD) is an acute febrile vasculitic syndrome of unknown etiology that preferentially affects the coronary artery. Interleukin-10 (IL-10) is a key proinflammatory cytokine, and a polymorphism near the major transcriptional start site of the IL-10 gene was shown to influence IL-10 production in vitro. This study investigated the association of the IL-10 promoter polymorphism with KD and its clinical parameters in Korean children. A total of 194 children with congenital heart disease (CHD) and 95 children with KD were included in this study. IL-10 (-627 A/C) polymorphism genotypes were determined using the single-base extension method. There was no difference in the allele frequencies of IL-10 (-627 A/C) polymorphism between CHD children and KD children. KD children with one or two copies of the IL-10 (-627C) allele showed significantly lower albumin levels (p = 0.020) and higher frequencies of early coronary artery aneurysm [62.22% versus 37.78%, adjusted odds ratio (aOR) = 3.50, 95% confidence interval (CI): 1.50–8.16] compared with KD children with the common IL-10 (-627A) allele. These findings suggest that the IL-10 (-627 A/C) promoter polymorphism might be a genetic marker for the risk of early coronary artery complication in KD.

Fabry disease: Biochemical, pathological and structural studies of the α-galactosidase A with E66Q amino acid substitution

Recently, male subjects harboring the c.196G>C nucleotide change which leads to the E66Q enzyme having low α-galactosidase A (GLA) activity have been identified at an unexpectedly high frequency on Japanese and Korean screening for Fabry disease involving dry blood spots and plasma/serum samples. Individuals with the E66Q enzyme have been suspected to have the later-onset Fabry disease phenotype leading to renal and cardiac disease. However, there has been no convincing evidence for this. To determine whether c.196G>C (E66Q) is disease-causing or not, we performed biochemical, pathological and structural studies. It was predicted that the E66Q amino acid substitution causes a small conformational change on the molecular surface of GLA, which leads to instability of the enzyme protein. However, biochemical studies revealed that subjects harboring the E66Q enzyme exhibited relatively high residual enzyme activity in white blood cells, and that there was no accumulation of globotriaosylceramide in cultured fibroblasts or an increased level of plasma globotriaosylsphingosine in these subjects. An electron microscopic examination did not reveal any pathological changes specific to Fabry disease in biopsied skin tissues from a male subject with the E66Q enzyme. These results strongly suggest that the c.196G>C is not a pathogenic mutation but is a functional polymorphism.