Constitutional haploinsufficiency of tumor suppressor genes in mentally retarded patients with microdeletions in 17p13.1.

Chromosome microdeletions or duplications are detected in 10-20% of patients with mental impairment and normal karyotypes. A few cases have been reported of mental impairment with microdeletions comprising tumor suppressor genes. By array-CGH we detected 4 mentally impaired individuals carrying de novo microdeletions sharing an overlapping segment of approximately 180 kb in 17p13.1. This segment encompasses 18 genes, including 3 involved in cancer, namely KCTD11/REN, DLG4/PSD95, and GPS2. Furthermore, in 2 of the patients, the deletions also included TP53, the most frequently inactivated gene in human cancers. The 3 tumor suppressor genes KCTD11, DLG4, and GPS2, in addition to the GABARAP gene, have a known or suspected function in neuronal development and are candidates for causing mental impairment in our patients. Among our 4 patients with deletions in 17p13.1, 3 were part of a Brazilian cohort of 300 mentally retarded individuals, suggesting that this segment may be particularly prone to rearrangements and appears to be an important cause (approximately 1%) of mental retardation. Further, the constitutive deletion of tumor suppressor genes in these patients, particularly TP53, probably confers a significantly increased lifetime risk for cancer and warrants careful oncological surveillance of these patients. Constitutional chromosome deletions containing tumor suppressor genes in patients with mental impairment or congenital abnormalities may represent an important mechanism linking abnormal phenotypes with increased risks of cancer.

Chromosome imbalances in syndromic hearing loss.

The cause of hearing impairment has not been elucidated in a large proportion of patients. We screened by 1-Mb array-based comparative genomic hybridization (aCGH) 29 individuals with syndromic hearing impairment whose clinical features were not typical of known disorders. Rare chromosomal copy number changes were detected in eight patients, four de novo imbalances and four inherited from a normal parent. The de novo alterations define candidate chromosome segments likely to harbor dosage-sensitive genes related to hearing impairment, namely 1q23.3-q25.2, 2q22q23, 6p25.3 and 11q13.2-q13.4. The rare imbalances also present in normal parents might be casually associated with hearing impairment, but its role as a predisposition gene remains a possibility. Our results show that syndromic deafness is frequently associated with chromosome microimbalances (14-27%), and the use of aCGH for defining disease etiology is recommended.

Genomic imbalances associated with mullerian aplasia.

BACKGROUND: Aplasia of the müllerian ducts leads to absence of the uterine corpus, uterine cervix, and upper (superior) vagina. Patients with müllerian aplasia (MA) often exhibit additional clinical features such as renal, vertebral and cardiac defects. A number of different syndromes have been associated with MA, and in most cases its aetiology remains poorly understood. OBJECTIVE AND METHODS: 14 syndromic patients with MA and 46,XX G-banded karyotype were screened for DNA copy number changes by approximately 1 Mb whole genome bacterial artificial chromosome (BAC) array based comparative genomic hybridisation (CGH). The detected alterations were validated by an independent method and further mapped by high resolution oligo-arrays. RESULTS: Submicroscopic genomic imbalances affecting the 1q21.1, 17q12, 22q11.21, and Xq21.31 chromosome regions were detected in four probands. Presence of the alterations in the normal mother of one patient suggests incomplete penetrance and/or variable expressivity. CONCLUSION: 4 of the 14 patients (29%) were found to have cryptic genomic alterations. The imbalances on 22q11.21 support recent findings by us and others that alterations in this chromosome region may result in impairment of müllerian duct development. The remaining imbalances indicate involvement of previously unknown chromosome regions in MA, and point specifically to LHX1 and KLHL4 as candidate genes.

Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents.

BACKGROUND: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. OBJECTIVE AND METHODS: Array-CGH with approximately 3500 large insert clones spaced at approximately 1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. RESULTS: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. CONCLUSIONS: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

Whole-genome array-CGH screening in undiagnosed syndromic patients: old syndromes revisited and new alterations.

We report array-CGH screening of 95 syndromic patients with normal G-banded karyotypes and at least one of the following features: mental retardation, heart defects, deafness, obesity, craniofacial dysmorphisms or urogenital tract malformations. Chromosome imbalances not previously detected in normal controls were found in 30 patients (31%) and at least 16 of them (17%) seem to be causally related to the abnormal phenotypes. Eight of the causative imbalances had not been described previously and pointed to new chromosome regions and candidate genes for specific phenotypes, including a connective tissue disease locus on 2p16.3, another for obesity on 7q22.1-->q22.3, and a candidate gene for the 3q29 deletion syndrome manifestations. The other causative alterations had already been associated with well-defined phenotypes including Sotos syndrome, and the 1p36 and 22q11.21 microdeletion syndromes. However, the clinical features of these latter patients were either not typical or specific enough to allow diagnosis before detection of chromosome imbalances. For instance, three patients with overlapping deletions in 22q11.21 were ascertained through entirely different clinical features, i.e., heart defect, utero-vaginal aplasia, and mental retardation associated with psychotic disease. Our results demonstrate that ascertainment through whole-genome screening of syndromic patients by array-CGH leads not only to the description of new syndromes, but also to the recognition of a broader spectrum of features for already described syndromes. Furthermore, on the technical side, we have significantly reduced the amount of reagents used and costs involved in the array-CGH protocol, without evident reduction in efficiency, bringing the method more within reach of centers with limited budgets.

Prevalence of the A1555G (12S rRNA) and tRNASer(UCN) mitochondrial mutations in hearing-impaired Brazilian patients.

Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNASer(UCN) gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%), which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNASer(UCN) did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190) of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNASer(UCN) gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern.

HIV type 1 infection of CD4+ T cells depends critically on basic amino acid residues in the V3 domain of envelope glycoprotein 120.

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a major determinant of phenotypic variability. The V3 domain of HIV-1 has many basic amino acid residues. Lymphocytotropic HIV-1 tends to have a V3 domain with a higher density of positive charge than does monocytotropic HIV-1. The importance of basic residues in the V3 domain for the HIV-1 infectivity, however, has not been well investigated. Here we show that mutation of basic amino acid residues at positions 303, 306, 309, 313, and 325 in the V3 domain of the lymphocytotropic isolate NL4-3 results in a dramatic elimination of both virus infectivity and syncytium-inducing ability. Three basic amino acid substitutions (at position 306, 309, and 313) induced a decrease in the binding ability of two kinds of neutralizing antibodies (NEA9284 and 0.5 beta) that recognize a different site in the V3 domain. This suggests that the basic residues play an important role in maintaining the tertiary structure of the V3 domain. Monocytotropism was not simply dependent on either decreased positive charge in the V3 domain of NL4-3 or on mutation of lysine to glutamate at position 320, which is a characteristic amino acid of monocytotropic HIV-1. These findings contribute to our understanding of the significance of basic residues on the function of envelope glycoprotein.

Heterozygosity probabilities for normal relatives of isolated cases affected by incompletely penetrant conditions and the calculation of recurrence risks for their offspring. I. Autosomal dominant genes.

Heterozygosity probabilities P(het) for relatives of isolated cases produced by incompletely penetrant autosomal dominant genes and recurrence risks for their offspring, R = P(het).K/2, where K is the penetrance value, have been calculated in the literature for some simple particular situations. Bayes theorem and elements from the theory of finite difference equations enabled us to derive the heterozygosity probability for any individual belonging to a pedigree containing an isolated case affected with an incompletely penetrant autosomal dominant disorder. The generalized formula here derived is valid for most particular cases thus far studied in the literature.

The importance of A'-d ridge count in dermatoglyphic diagnosis of the Ullrich-Turner syndrome.

The dermatoglyphic characteristics of 52 women with the Ullrich-Turner syndrome were studied and compared to those of a control group of 50 normal women. Through the use of discriminant analysis, it is shown that the use of the A'-d ridge count alone has the same efficiency in separating Ullrich-Turner syndrome patients from normal female subjects as discriminant functions here derived and other methods already published in the literature.

Evaluation of carrier detection rates for Duchenne and Becker muscular dystrophies using serum creatine-kinase (CK) and pyruvate-kinase (PK) through discriminant analysis.

Serum pyruvate-kinase (PK) and creatine-kinase (CK) determinations have been carried out in a sample of 100 obligate carriers for the Duchenne muscular dystrophy (DMD) gene, 23 obligate carriers for the Becker muscular dystrophy (BMD) gene, and 50 normal adult control women. Blood samples were collected from all subjects three times on three independent occasions and the means of these three determinations were considered for both PK and CK activities in the statistical analysis. Discriminant analysis has shown that, in the group of carriers for the DMD gene, the estimated misclassification frequencies (M.F.) using either serum CK, PK, or both enzymes were: 26.5% for CK alone, 19.5% for PK alone, and 19% for both enzymes. In the group of carriers for the BMD gene, the estimated proportions of M.F. were: 31.7% for CK alone, 23.8% for PK alone, and 20.4% for both enzymes. It is concluded that, although a proportion of carries still remains undetected, the use of serum PK determinations enhances the capability of detecting carriers of both DMD and BMD mainly when compared with serum CK alone.

Serum CK-MB activity in progressive muscular dystrophy: is it of nosologic value?

Serum creatine-kinase (CK) isoenzyme MB was measured in 53 patients affected by different types of myopathies (20 with Duchenne muscular dystrophy (DMD), eight with the Becker form (BMD), ten with the limb-girdle form (LGMD), six with the facioscapulohumeral form (FSH), and nine affected by polymyositis and in 21 normal control subjects). The aim of this study was to compare each group with the control individuals and to assess the nosologic value of CK-MB activity among some clinically similar dystrophies, which may have an important application for genetic counseling. A statistically significant increased CK-MB activity was found only in the Duchenne and Becker patients when compared with control persons (p less than 0.05). When the different groups of patients were compared among themselves, no significant difference was found between DMD and BMD or LGMD and polymyositis. However, a significant difference was found between BMD and LGMD. Based on these data, it is possible, through discriminant analysis, to estimate the relative biochemical probability of an isolated male patient belonging to either group.

Notes on the population genetics of fragile X syndrome.

Our analysis of fragile X-inactivation in normal and mentally retarded heterozygotes led us to conclude that a fraction of female carriers of the imprinted (fully mutated) allele is phenotypically normal as a consequence of X-inactivation. Taking this into account, we derived equilibrium equations for the fragile X [fra(X)] genotype frequencies. We also showed that small variations in the value of s (selection coefficient of affected heterozygotes) and r (imprinting rate during oogenesis) affect genotype ratios significantly.

Unusual type of brachydactyly associated with short stature and facial anomalies. A new syndrome?

We report on a 10-year-old boy with short stature, mild microcephaly, malar hypoplasia, highly arched palate, prominent upper incisors, micrognathia, and unusual digital anomalies involving the proximal phalanges of fingers 2-5 of both hands. To our knowledge, this is a hitherto undescribed syndrome.

Calculation of recurrence risks for heterogeneous genetic disorders.

We present general formulae for several common situations in the genetic counseling of heterogeneous disorders. The occurrence or not of parental consanguinity is taken into account, since it distorts significantly the prior probabilities favoring the different mechanisms. Nonsyndromic deafness is used as a numerical application, since it can be produced by any type of monogenic inheritance and can be mixed with variable proportions of environmental cases. Recurrence risks are calculated including or not including environmental factors in the origin of the defect. In underdeveloped countries the proportion of environmentally determined cases of deafness is significantly higher than in first-world countries. Therefore, when environmental causes cannot be excluded, recurrence risks are always higher in developed than in developing countries. On average, when parental consanguinity is present there is a significant increase in recurrence risks for deafness, whether environmental factors are included or not.

Effect of X inactivation on fragile X frequency and mental retardation.

The probability of a heterozygote being affected was estimated from the distribution of frequencies of early-replicating fragile X [fra(X)] chromosome in normal and mentally retarded heterozygotes, taking into account the prior probabilities of 0.35 for mental retardation and 0.65 for normality. The estimated probability of a heterozygote with 100% early-replicating fra(X) being mentally retarded was 78%, which coincides with the value of penetrance in males. Therefore, the manifestation of retardation in females seems to differ from that in males due solely to X inactivation. The frequencies of early-replicating fra(X) were significantly increased among the heterozygotes with the highest frequencies of fra(X) both in the normal group and in the mentally retarded. The mean frequencies of early-replicating fra(X) were 0.42 and 0.68 for normal and mentally retarded heterozygotes, respectively. Considering the overall frequency of retarded heterozygotes as 0.35, the mean frequency of early-replicating fra(X) obtained for all heterozygotes was 0.51, which is in accordance with the hypothesis of random X inactivation. Thus the fragile site appears to have equal chances of being detected when located either on the early- or on the late-replicating X. This leads to the conclusion that the frequency of the fragile site is a consequence of the proportion of cells with the active Martin-Bell syndrome (MBS) gene and not the result of a better visualization of the site on the early-replicating X.

Relationship between Mayer-Rokitansky-Küster (MRK) anomaly and hereditary renal adysplasia (HRA).

We report the results of a study performed in a sample of women with the Mayer-Rokitansky-Küster (MRK) anomaly and in their first-degree relatives. Our results are compatible with a traditional model of multifactorial determination; however, we cannot exclude the hypothesis of autosomal dominant inheritance, with an intermediate degree of penetrance and a highly variable expressivity of a single mutant gene. In this sense, our data seem to support the idea expressed recently by Opitz [1987].

Absence of correlation between skewed X inactivation in blood and serum creatine-kinase levels in Duchenne/Becker female carriers.

The pattern of X inactivation in lymphocyte DNA was investigated in 107 Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) carriers (102 asymptomatic and 5 manifesting carriers) and 117 normal female controls of different ages, with the aim: a) to analyze the pattern of X inactivation in blood DNA of a large number of DMD/BMD carriers as compared to normal female controls; b) to determine if there is a decrease in serum creatine kinase (CK) levels with age in obligate DMD/BMD carriers; c) to determine if there is a correlation between X-chromosome inactivation and serum CK among asymptomatic DMD/BMD carriers of different ages or with different clinical manifestations in symptomatic carriers. A high proportion of females showed extremely skewed X inactivation (>90% of one X preferentially inactivated), which was almost the same among carriers and normal controls (19 and 24%, respectively). The mean serum CK was significantly greater among young (<20 years old) than adult (>20 years old) DMD/BMD carriers and it decreased significantly until age 20 with an apparent stabilization afterwards. No statistically significant correlation was found between the proportion of active X(DMD) in blood and serum CK activity in DMD/BMD carriers although it was higher among those less than 20 years old. Our observations suggest that highly skewed X-chromosome pattern in blood (with preferential inactivation of the X(N) chromosome) is not enough to predict that a young DMD carrier will develop muscular weakness.

Effect of mazindol on growth hormone levels in patients with Duchenne muscular dystrophy.

Human growth hormone (HGH) inhibition may be beneficial in Duchenne muscular dystrophy (DMD) and slow down the rate of progression of the disease. The purposes of the present investigation were: 1) to assess, through pharmacological stimuli (L-dopa test), the HGH response in untreated DMD patients, and 2) to evaluate the inhibitory effect of mazindol on HGH levels as a potential treatment for DMD. In 55 DMD patients, HGH levels were measured through the L-dopa test, and 40 received mazindol. After 1 year, there was wide variability in the individual response to mazindol. An apparent diminution in the mean HGH level was observed in the whole group of patients; this was statistically significant after 3 and 6 months but not after 9 and 12 months of treatment. The results suggest that this drug is not effective for arresting growth or inhibiting HGH secretion for a prolonged period of time.

Analysis of a novel functional polymorphism within the promoter region of the serotonin transporter gene (5-HTT) in Brazilian patients affected by bipolar disorder and schizophrenia.

It has been suggested that the serotonin transporter (5-hydroxytryptamine-transporter or 5-HTT) may be involved in the pathogenesis of affective disorders. Recently, Collier et al. (1996) found that the frequency of the low-activity short variant (s) of the 5-HTT-linked polymorphic region (5-HTTLPR) was higher among patients with affective disorders than in normal controls. However, since the observed level of significance was not high, they suggest that these findings should be replicated in independent samples. We have analyzed 86 unrelated patients (47 with bipolar disorder and 39 with schizophrenia) and 98 normal controls from the Brazilian population for the 5-HTTLPR. Statistical analysis revealed that the genotypes (LL, Ls, ss) as well as the estimated allele frequencies (L,s) did not differ significantly among the three studied groups or between bipolar and normal controls. In addition, although not statistically significant, the genotype ss in our sample was less frequent among our bipolar patients than in our normal controls (12.8% versus 16.3%) which is the opposite of what was found by Collier et al. (24% versus 18%) in the European study. Although it will be important to extend the present analysis in a larger sample, our preliminary results suggest that the 5-HTTLPR does not seem to play a major role in the genetics of bipolar and schizophrenic disorders at least in this group of Brazilian psychiatric patients.