Silver-Russell syndrome: genetic basis and molecular genetic testing

Hereditary combined vitamin K-dependent clotting factors deficiency (VKCFD) is a rare congenital bleeding disorder resulting from variably decreased levels of coagulation factors II, VII, IX and X as well as natural anticoagulants protein C, protein S and protein Z. The spectrum of bleeding symptoms ranges from mild to severe with onset in the neonatal period in severe cases. The bleeding symptoms are often life-threatening, occur both spontaneously and in a surgical setting, and usually involve the skin and mucosae. A range of non-haemostatic symptoms are often present, including developmental and skeletal anomalies. VKCFD is an autosomal recessive disorder caused by mutations in the genes of either gamma-glutamyl carboxylase or vitamin K2,3-epoxide reductase complex. These two proteins are necessary for gamma-carboxylation, a post-synthetic modification that allows coagulation proteins to display their proper function. The developmental and skeletal anomalies seen in VKCFD are the result of defective gamma-carboxylation of a number of non-haemostatic proteins. Diagnostic differentiation from other conditions, both congenital and acquired, is mandatory and genotype analysis is needed to confirm the defect. Vitamin K administration is the mainstay of therapy in VKCFD, with plasma supplementation during surgery or severe bleeding episodes. In addition, prothrombin complex concentrates and combination therapy with recombinant activated FVII and vitamin K supplementation may constitute alternative treatment options. The overall prognosis is good and with the availability of several effective therapeutic options, VKCFD has only a small impact on the quality of life of affected patients.     

Quality control in molecular genetic testing

DNA-based testing for genetic diseases has developed from nothing into a principal part of laboratory medicine over the past 15 years. In the rush to bring these powerful new technologies into medical use, issues of quality have not always been given sufficient attention. Efforts are now being made to assess the quality of the output of genetic testing laboratories, and the results show that there is room for improvement.     

Protein molecular function influences mutation rates in human genetic diseases with allelic heterogeneity

Molecular epidemiology studies have used the counts of different mutational types like transitions, transversions, etc. to identify putative mutagens, with little reference to gene organization and structure–function of the translated product. Moreover, geographical variation in the mutational spectrum is not limited to the mutational types at the nucleotide level but also have a bearing at the functional level. Here, we developed a novel measure to estimate the rate of spontaneous detrimental mutations called “mutation index” for comparing the mutational spectra consisting of all single base, missense, and non-missense changes. We have analyzed 1609 mutations occurring in 38 exons in 24 populations in three diseases viz. hemophilia B (F9 gene – 420 mutations in 9 populations across 8 exons), hemophilia A (F8 gene – 650, 8 and 26, respectively) and ovarian carcinoma (TP53 gene – 539, 7 and 4, respectively). We considered exons as units of evolution instead of the entire gene and observed feeble differences among populations implying lack of a mutagen-specific effect and the possibility of mutation causing endogenous factors. In all the three genes we observed elevated rates of detrimental mutations in exons encoding regions of significance for the molecular function of the protein. We propose that this can be extended to the entire exome with implications in exon-shuffling and complex human diseases.     

High allelic heterogeneity between Afro-Brazilians and Euro-Brazilians impacts cystic fibrosis genetic testing

Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 1,000 different mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). To determine the frequency of 70 common worldwide CFTR mutations in 155 Euro-Brazilian CF patients and in 38 Afro-Brazilian CF patients, we used direct PCR amplification of DNA from a total of 386 chromosomes from CF patients born in three different states of Brazil. The results show that screening for seventy mutations accounts for 81% of the CF alleles in Euro-Brazilians, but only 21% in the Afro-Brazilian group. We found 21 different mutations in Euro-Brazilians and only 7 mutations in Afro-Brazilians. The frequency of mutations and the number of different mutations detected in Euro-Brazilians are different from Northern European and North American populations, but similar to Southern European populations; in Afro-Brazilians, the mix of CF-mutations is different from those reported in Afro-American CF patients. We also found significant differences in detection rates between Euro-Brazilian (75%) and Afro-Brazilian CF patients (21%) living in the same state, Minas Gerais. These results, therefore, have implications for the use of DNA-based tests for risk assessment in heterogeneous populations like the Brazilians. Further studies are needed to identify the remaining CF mutations in the different populations and regions of Brazil.     

Clinical and molecular genetic features of ARC syndrome

Arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome (MIM 208085) is an autosomal recessive multisystem disorder that may be associated with germline VPS33B mutations. VPS33B is involved in regulation of vesicular membrane fusion by interacting with SNARE proteins, and evidence of abnormal polarised membrane protein trafficking has been reported in ARC patients. We characterised clinical and molecular features of ARC syndrome in order to identify potential genotype-phenotype correlations. The clinical phenotype of 62 ARC syndrome patients was analysed. In addition to classical features described previously, all patients had severe failure to thrive, which was not adequately explained by the degree of liver disease and 10% had structural cardiac defects. Almost half of the patients who underwent diagnostic organ biopsy (7/16) developed life-threatening haemorrhage. We found that most patients (9/11) who suffered severe haemorrhage (7 post biopsy and 4 spontaneous) had normal platelet count and morphology. Germline VPS33B mutations were detected in 28/35 families (48/62 individuals) with ARC syndrome. Several mutations were restricted to specific ethnic groups. Thus p.Arg438X mutation was common in the UK Pakistani families and haplotyping was consistent with a founder mutation with the most recent common ancestor 900–1,000 years ago. Heterozygosity was found in the VPS33B locus in some cases of ARC providing the first evidence of a possible second ARC syndrome gene. In conclusion we state that molecular diagnosis is possible for most children in whom ARC syndrome is suspected and VPS33B mutation analysis should replace organ biopsy as a first line diagnostic test for ARC syndrome.     

Rett syndrome molecular diagnosis and implications in genetic counseling

Rett syndrome is a rare genetic X-linked dominant disorder. This syndrome is the most frequent cause of mental retardation in girls. In the classical form of the disease, the presenting signs and the course of development are characteristic. However clinical diagnosis can be very difficult when the expression is not in the classical form. Mutations in MeCP2 are responsible for 80% of cases. When MeCP2 mutation is found in an index case, genetic counseling is similar to that in other X-linked dominant genetic diseases. However, mutations in this gene can cause a spectrum of atypical forms. On the other hand, other genetic conditions like translocations, sex chromosome numerical anomalies, and mutations in other genes can complicate genetic counseling in this syndrome. We present the first case of molecular diagnosis of Rett syndrome in Iran and discuss the recent developments in its genetic counseling.     

Results of dynamic endocrine testing of hypothalamic pituitary function in patients with a primary "empty" sella syndrome

A total of 52 patients (38 women, 14 men) with a primary "empty" sella syndrome underwent dynamic endocrine testing consisting of insulin-induced hypoglycaemia and a combined anterior pituitary stimulation test utilizing GnRH and TRH. The diagnosis of an "empty" sella turcica was made on the basis of thin collimation CT reconstructions and in addition either metrizamide cisternography or magnetic resonance (MR) imaging. Only 16 of the patients presented with endocrine problems. Hyperprolactinaemia was the most common endocrine disturbance detected, and was found in 17 individuals. Panhypopituitarism was found in 3 patients. Nine other patients had some degree of partial pituitary insufficiency, but only two of them required replacement therapy. However, 31 patients had no evidence of endocrine dysfunction.     

Lower gastrointestinal disorders in patients with irritable bowel syndrome

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal system characterized by abdominal pain related to bowel emptying, defecation impairment and abdominal distention. The aim of the study was to objectify lower gastrointestinal system disturbances in IBS patients. Thirty IBS patients and 30 healthy subjects were included in the study. IBS patients were divided into two subgroups: IBS with predominant diarrhea (IBSd) and IBS with predominant constipation (IBSc). All study subjects underwent physical examination (including digitorectal examination), standard laboratory testing and anorectal manometry. Endoscopy was performed only in group of IBS patients. A statistically significant difference was recorded in most manometric parameters between healthy subjects and IBS patients, which was even more pronounced in IBSd patients. Study results showed that the intestinal motility disorder underlying IBS could be objectified by use of anorectal manometry.     

禽源多重耐药金黄色葡萄球菌耐药基因检测及分子分型

【背景】金黄色葡萄球菌是革兰氏阳性菌,在动物和人身上能引起一系列疾病。【目的】了解安徽省不同地区禽源多重耐药金黄色葡萄球菌耐药性的情况及基因分型特征。【方法】以安徽不同地区的病禽肝脏作为标本,分离鉴定得到103株多重耐药金黄色葡萄球菌,并进行耐药基因型检测和ERIC-PCR分子分型。【结果】耐药菌株从三重到八重耐药均有分布,主要集中在五重(43/103)、四重(21/103)和六重耐药(22/103)。药敏结果显示,β-内酰胺类的耐药率最高(79.6%),氨基糖苷类次之(71.8%)。耐药基因检出率由高到低分别为mec A(92.2%)、aac(6′)/aph(2″)(76.7%)、ermC(37.9%)、ermA(13.6%)和fem A(3.9%)。ERIC-PCR分子分型获得6种不同类群,优势流行菌群为类群Ⅱ(38/103)。【结论】安徽地区金黄色葡萄球菌存在较严重的耐药性,氨基糖苷类、β-内酰胺类和大环内酯类抗生素的耐药基因携带率较高。分型结果表明安徽部分区域耐药金黄色葡萄球菌具有遗传多样性,但耐药谱与ERIC-PCR分子分型无明显关联。     

Discovery of allelic variants of HOXA1 and HOXB1: genetic susceptibility to autism spectrum disorders

BACKGROUND: Family studies have demonstrated that the autism spectrum disorders (ASDs) have a major genetic etiologic component, but expression and penetrance of the phenotype are variable. Mice with null mutations of Hoxa1 or Hoxb1, two genes critical to hindbrain development, have phenotypic features frequently observed in autism, but no naturally occurring variants of either gene have been identified in mammals. METHODS: By sequencing regions of genomic DNA of patients with autism spectrum disorders, we detected a substitution variant at HOXA1 and an insertion variant at HOXB1, both in coding regions of the genes. Fifty-seven individuals ascertained for a diagnosis of an ASD, along with 166 of their relatives, were typed for these variants. Two non-ASD populations were typed, and the frequency of the newly identified alleles was determined in all groups. The genotypes of the ASD families were tested for conformation to Hardy-Weinberg proportions and Mendelian expectations for gene transmission. RESULTS: The frequency of the variants was 10-25% in persons of European or African origin. In the ASD families, there was a significant deviation from the HOXA1 genotype ratios expected from Hardy-Weinberg proportions (P = 0.005). Among affected offspring, a significant deviation from Mendelian expectation in gene transmission (P = 0.011) was observed. No statistically significant effects were detected when the same analyses were applied to the HOXB1 locus, but there was evidence of an interaction between HOXA1, HOXB1, and gender in susceptibility to ASDs. CONCLUSIONS: The results support a role for HOXA1 in susceptibility to autism, and add to the existing body of evidence implicating early brain stem injury in the etiology of ASDs.     

Clinical genealogical and molecular genetic study of patients with mental retardation

The results of clinical, genealogical, cytogenetic and molecular genetic studies of 113 patients from 96 families with different forms of mental retardation from Ukraine are presented. This study was held as part of the CHERISH project of the 7-th Framework Program. The aim of the project is to improve diagnostics of mental retardation in children in Eastern Europe and Central Asia through detailed analysis of known chromosomal and gene's aberrations and to find the new gene-candidates that cause mental retardation. All patients have normal chromosome number (46XY or 46XX). The cases with fragile-X syndrome were eliminated using molecular genetic methods. Genome rearrangements were found among 28 patients using cytogenetic analysis, multiplex ligation-dependent probe amplification (MLPA analysis) ofsubtelomeric regions and array-based comparative genomic hybridisation (array CGH screening). In 10 cases known pathogenic CNV's were identified, 11 cases are unknown aberrations; their pathogenicity is being determined. The rest cases are known nonpathogenic gene rearrangements. Obtained results show the strong genetic heterogeneity of hereditary forms of mental retardation. The further studies will allow to identificate genes candidates and certain mutations in these genes that may be associated with this pathology.     

Clinical, biochemical and molecular genetic data in five children with Gitelman's syndrome.

Gitelman's variant of Bartter's syndrome, inherited hypokalemic alkalosis, is caused by mutation in the thiazide-sensitive NaCl co-transporter (NCCT). The main clinical symptoms are: muscular weakness, carpopedal spasm, constipation and short stature. The diagnosis was suspected in five children according to clinical criteria. All patients exhibited carpopedal spasm during febrile illness, three patients had short stature. Biochemical features were: metabolic alkalosis, hypokalemia, hypomagnesemia, low IGF-I levels, hyperkaliuria, hypernatriuria, hypocalciuria and normoprostaglandinuria. Three patients had elevated plasma renin activity and hyperaldosteronism. Mutational analysis of the NCCT gene confirmed the diagnosis in all five patients. Different forms of therapy, potassium and magnesium substitution, spironolactone and indomethacin failed to fully correct hypokalemia and hypomagnesemia, but markedly improved growth velocity and normalized IGF-I levels in the three patients with short stature. During therapy, clinical symptoms disappeared. We conclude that Gitelman's syndrome is a disorder with a variable symptom profile, but can be suspected on clinical signs already in early childhood. The early diagnosis is essential in preventing complications.     

Designing and testing scene enhancement algorithms for patients with retina degenerative disorders

Background  

Retina degenerative disorders represent the primary cause of blindness in UK and in the developed world. In particular, Age Related Macular Degeneration (AMD) and Retina Pigmentosa (RP) diseases are of interest to this study. We have therefore created new image processing algorithms for enhancing the visual scenes for them.     

Frequencies of allelic polymorphism of endothelial NO-synthase gene in patients with acute coronary syndrome in Ukrainian population

Endothelial NO-synthase (eNOS) gene allelic polymorphism in 221 patients with acute coronary syndrome and in 83 practically healthy people was determined. It was shown that interrelations of normal homozygotes, heterozygotes and pathologic homozygotes in T/C promoter polymorphism analysis accout 48%, 36% and 16% correspondingly (in control--48%, 46%, 6%; P < 0.05 by chi2-test); in G894 --> T polymorphism ofexon 7 analysis--34%, 58%, 8% (in control--29%, 67%, 4%; P > 0.05), and in determination of 4a/4b polymorphism of intron 4--64.5%, 31% and 4.5% (in control--62.5%, 32.5%, 5%; P > 0.05). Obtained data show that eNOS C/C promoter variant is a risk factor of acute coronary syndrome in Ukrainian population.