Pediatric Fragile X Chromosome

Introduction to fragile X chromosome in children Fragile X chromosome, also known as fragile X syndrome (fragileXsyndrome, FXS), is an incompletely explicit X-linked allogenic hereditary disease, because the short arm Xq27.3 of the patient's X chromosome has a brittle fracture. Named after the point. FXS is a familial mental disorder with clinical manifestations of mental retardation, special face, giant testis, large ears, and abnormal language and behavior. basic knowledge The proportion of sickness: 0.0052% Susceptible people: children Mode of infection: non-infectious Complications: epilepsy

Childhood fragile X chromosome cause

(1) Causes of the disease

The occurrence of the fragile site at the end of the X chromosome may be related to deoxythymidine-phosphate deficiency during DNA anabolism, while the fragile site is a DNA-rich segment. When deoxythymidine-phosphate is reduced, deoxythymidine The triphosphate is reduced so that this segment does not fold tightly during mitosis, and even cracks or breaks appear to exhibit brittleness.

Previous studies on fragile X chromosomes have mostly focused on cytogenetic levels. With the deepening of molecular biology research and the discovery of some special genetic laws in the disease, the classification of fragile parts, the special inheritance of fragile X chromosomes. Many important advances have been made in the research on its mechanism of production, and some new concepts and theories have been formed to enable people to understand the disease from a new level and perspective.

Fragile part classification: 1 hereditary brittle part (h-fra), also known as rare brittle part; 2 structural type brittle part (c-fra), also known as common type brittle part.

The genetic characteristics of the fragile X chromosome: In the past, its typical genetic pattern was X-linked recessive inheritance. In recent years, its genetic pattern is very complicated, and it has a special genetic law that is completely different from the general genetic disease: 1 is through males without abnormal phenotype Carriers, also known as the explicit (NP) transmission, have no abnormalities in their fragile X daughters; 2 in the fra(X) family, the mentally retarded male patients account for about 20%, the isolation rate is 0.4; 3 phenotypic abnormal brittleness Among the sons born to X women, the isolation rate is 0.5; the fragility X of 4 phenotypic women is from their mother, not from the father; 5 about 35% of female carriers have mental retardation; 6NP male fragile X mother general phenotype All were normal, and the risk of phenotypic abnormality in patients with NP males was lower; 7 almost all mothers with fragile X syndrome had brittle X; 8 had different degrees of appearance in siblings.

Several hypotheses about the production mechanism: 1 movable factor insertion hypothesis; 2 poly-purine/polypyrimidine sequential amplification hypothesis; 3 pyrimidine-rich DNA sequence recombination and amplification hypothesis; 4 pre-stability pre-mutation hypothesis; Chromosome repression gene effect hypothesis.

Jacobs et al believe that the genes of male and female germplasm cytoplasm can have the same mutation frequency, and all are transmitted downwards. Patients with fragile X chromosome may be passed down to the parent or mother through two stages of genetic engineering mutation, but the parent or mother phenotype Normally, fra(X) is often not detected in the peripheral blood, and when his or her germ cells are once again mutated and passed to the child or female, a fra(X)-positive patient or female carrier is produced, Sutherland It has been pointed out that there are no abnormal gene products or lack of certain products in patients with fragile X chromosome. There is no basis at present, and the true mechanism of occurrence of fragile sites is still to be further studied.

(two) pathogenesis

The fragile site (FRAXA) in the Xq27.3 region of patients with fragile X chromosome is a typical cytogenetic feature. In 1993, the fragile X chromosome coding gene cDNA was cloned, and the n copy number in the (CGG)n structure was found to be normal. When 52 is amplified to 230, it is the molecular basis of the pathogenesis of patients with fragile X chromosome. The abnormally amplified (CGG)n structure is located in exon 1 of the translational region of FMR-I gene. So far, missense mutations and deletions of FMRI gene are known. Mutant carriers also exhibit the same clinical symptoms of dynamic mutations in the FMRI gene, indicating that patients with clinical syndromes of fragile X chromosome have a high degree of genetic heterogeneity, further complicating the genetic diagnosis of these patients and their family members.

1. Cytogenetics: The X chromosome long arm 27.3 carries a folate-sensitive part associated with the fragile X chromosome. This part can be shown as a brittle fracture after special treatment, so it is called a fragile part, and the chromosome with a fragile part is called Fragile chromosomes have been found in the fragile parts of 26 chromosomes. Only the fragile part of the X27-X28 region of the X chromosome (FRAXA) is associated with hereditary diseases, while other fragile parts unrelated to the disease are called common fragile parts.

The mechanism of X-brittle sites is not fully understood. It is currently thought to be related to the anabolic process of DNA. It has been found that treatment with cytosolic acid or a higher dose of 5-fluorouracil (5-FU) can cause thymidine nucleosides. The synthetic part is inhibited, and the chromosome structure may cause cracks or breaks at specific parts.

2. Structure, transcription and translation of the FMR-I gene: The gene for the fragile X chromosome is called fragile X mental retardation-I (FMR-I), which is located in the Xq27.3 region, in the genome. It spans 38kb and consists of 17 exons and 16 introns. The mRNA of FMR-I gene is 4.4kb, encoding a brittle intelligent backward protein (FMRP) with a molecular weight of 69-70kDa and 596 amino acids. This is an RNA binding protein that is expressed in a variety of tissues in the body.

The exon of FMR-I gene is small (51-196 bp), but the intron is larger, the average size is 2.2 kb, and the intron 1 is about 9.9 kb. There are many kinds of FMR-I gene involved in the gene. Multiple transcriptional splicing forms of exons 10, 12, 14, 15 and 17, in which exons 12 and 14 are involved, usually result in the loss of the entire exon, but only those involving exons 10, 15 and 17 The partial sequence at the 5' end of these three exons is lost, because there is a concatenated conserved signal on the 5' end of each of the three exons, and the post-transcriptional splicing occurs at this position, resulting in these three The partial sequence at the 5' end of the exon is lost.

3. Dynamic mutation of FMR-I gene: There is a CGG trinucleotide repeat region at the 5' end of FMR-I gene. The number of repeats of CGG structure in normal individuals is polymorphic, ranging from 6 to 52 times, with an average of Thirty times, the most common (CGG)28 in the Chinese population, in the FXS patients, the CGG copy number is generally >200, and the more than 1000 times. The root cause of the fragile X chromosome is the mutation of the FMR-I gene. Therefore, dynamic mutation refers to the instability of CGG copy number during FMR-I gene transfer, which is the molecular genetic basis of more than 95% of FXS patients. Dynamic mutations include three types:

(1) Premutation of FMR-I gene: When the n-copy number of the FMR-I gene (CGG) n structure is amplified to 53-230, although the phenotype is normal, the carrier is prone to further in the process of passage. Amplification, the CGG repeat number of the offspring is greatly increased, and an abnormal phenotype appears. This mutation of the FMR-I gene is called a pre-mutation, and the intelligence level of the male or female FMR-I pre-mutation gene carrier is normal. There is no difference.

According to statistics, there is no difference in n copy number in male or female pre-mutant FMR-I gene (CGG) n structure, but with the gradual n-copy number in the FMR-I gene (EGG) n structure carried by women Increasing, the probability of amplification before amplification is gradually increased, and the n-copy number of the (CGG) structure is reduced when 38% of the pre-mutant FMR-I gene is transferred from father to daughter. The phenomenon is only seen when 2% of the maternal pre-mutant FMR-I gene is transmitted to the daughter, suggesting that the pre-mutant FMR-I gene has an amplification of the n-copy number in the (CGG)n structure during mother-to-female transmission. Tendency, but there is a tendency to shrink when the father and the daughter pass.

(2) Full mutation of FMR-I gene: When the FMR-I gene is amplified from 53 to 230 times before the pre-mutation state (CGG) to >230 times, 100% of male carriers exhibit typical fragile X synthesis. 53% of female carriers showed mental retardation ranging from mild to severe, which is called full mutation at this time. The whole mutation is directly related to the appearance of mental retardation. It was found that when the number of CGG structures was repeated 230 times Above, the CpG island at the 5' end of the FMR-I gene began to be abnormally methylated, and this methylation extended to the promoter region, resulting in inability to initiate transcription, mRNA not transcribed, and lack of protein products encoded by the gene. Causes clinical symptoms.

It is worth noting that a very small number of male fully-mutant FMR-I gene carriers lack the fragility of FRAXA sites, and the molecular genetics basis needs further study. In terms of mental retardation, almost 100% of males FMR-I full mutation gene carriers have mental retardation, of which about 89% (245/274) are moderately mentally retarded, but only 21% (36/170) of female FMR-I full mutation carriers exhibit moderate mental retardation. And up to 59% (100/170) of female FMR-I full mutant carriers do not appear mentally retarded.

(3) Back-mutation of the FMR-I gene: The CGG structure of the FMR-I gene in the pre-mutation or full-mutation state undergoes a certain range of reduction in the number of copies during the passage, which is called the back mutation of the FMR-I gene ( Reverse mutation), depending on the state of the FMR-I gene before and after the mutation, the back mutation can be divided into three types: full mutation premutation; full mutation full mutation or premutation chimerism; chimeric or Pre-mutant normal FMR-I gene, these phenomena can occur in the process of paternal transmission, but also in the process of maternal transmission, although less common, it increases the difficulty of predicting the dynamic mutation of FMR-I gene This has led to further complications in family genetic counseling and prenatal genetic diagnosis.

4. The influence of gender factors on the dynamic mutation of FMR-I gene: It is considered that the amplification of FMR-I gene (CGG) n structure is a multi-path multi-step amplification process, and the genetic pattern of fragile X chromosome often has special The law, that is, a male carrier with normal phenotype can pass the fragile part to his daughter, who generally has no mental retardation or other clinical symptoms, but she can pass the affected chromosome to the offspring, so that the third generation in the family appears. In FXS patients, the third-generation boys have more obvious mental retardation, and girls often have no obvious mental abnormalities, that is, the harm caused by the mother to the children is greater than that of the father, and the boy is more affected than the girl. This phenomenon is called the Sheman phenomenon. In addition, it is also found that only the pre-mutant FMR-I gene exists in the sperm sample of the male full-mutant FMR-I gene carrier. Therefore, it is considered that the full mutation of the FMR-I gene does not affect the male whole. The germ cells of the carrier are mutated, but there is currently insufficient evidence to suggest that the female egg has not undergone a full mutation of the FMR-I gene.

In addition, during the passage of female FMR-I gene mutation carriers, the expansion and size of the (CGG)n structure changes according to the sex of the offspring, that is, it has a tendency to further expand when passed to male offspring. However, when it is passed to female offspring, the degree of expansion is small, and the structure of (CGG)n is reduced. It is possible that another normal FMR-I gene carried by female X chromosome inhibits the early stage of female embryo. Further amplification of the fully-mutant FMR-I gene (CGG) n structure, in summary, the trend of the full-mutant FMR-I gene (CGG) n structure (either amplification or reduction) and moderately variable size are still affected by the parent And the dual influence of the offspring gender, therefore, this mutational feature of the FMR-I gene must be considered in the genetic counseling of the FMR-I gene dynamic mutation family.

5. Non-dynamic mutation of FMR-I gene: In addition to dynamic mutation of FMR-I gene, non-dynamic mutations such as base substitution and deletion occur in a few patients. One missense mutation and eight deletion mutations have been found. These mutations cause the same clinical symptoms and dynamic mutations, but lack the FRAXA fragile site. The current report does not show that this mutation has a hot spot.

Fragile X chromosome prevention in children

Because the FMRI gene has been confirmed as the basis of the fragile X chromosome, a more specific and low-cost blood test (DNA test for fragile X) has been established, and CGG can be detected in the blood of the whole mutation and the former mutant. The rate of expansion of nucleotide repeats, this test can be used for prenatal diagnosis, and experts recommend this method for all patients with unexplained mental retardation.

In 1995, a method for detecting FMRP and detecting blood antibodies to fully mutated individuals was established, which made it possible to screen for fragile X chromosome newborns.

Fragile X chromosome complications in children Complications

Mental retardation, growth disorders, mental and psychological disorders, epilepsy, etc.

Children's fragile X-chromosome symptoms Common symptoms Learning difficulties Language developmental anxiety Anxiety bowel dysarthria Fingerlet single pleat Depression Breathing ear position low

1. Male patients are characterized by low intelligence, large ears and large testicles. Typical clinical symptoms include:

(1) Mental retardation: IQ is often lower than 50, and is progressively intensified.

(2) Special face: The birth weight is relatively high, and the growth rate is fast in the first few years after birth, but the body is short, the face is slender, the forehead is prominent, the head circumference is enlarged, the sputum is full, the iris color is light, and the ear is everted. , high bow, big mouth, thick lips and large jaw and prominent.

(3) Large testicles: more testicular enlargement in prepuberty, testicular up to 30 ~ 50cm in late youth, thickening of the scrotum, rare in young patients, often accompanied by large penis.

(4) Language developmental disorders: more common, characterized by severe developmental delays in conversational and verbal expression, dysarthria, pathological imitation and repetitive speech, and lack of grammar and vocabulary.

(5) Abnormal personality behavior: including active, lack of concentration, temperament, strong rebellious psychology, anxiety and self-harm.

(6) nervous system symptoms: more mild, common for limb dyskinesia, involuntary movement retardation, excessive joint stiffness and systemic hyperreflexia, occasionally convulsions.

(7) Reproductive system: low sexual function, adult female pubic hair is female distribution and feminine breast, but can give birth to offspring.

2. Female carriers: may present mild mental retardation, premature ovarian failure, 70% of female carriers have pre-mutation without significant physiological or cognitive, abnormal behavior, while the other 30% have full mutations Women will show a range of symptoms. In general, women with symptoms have only slight changes in appearance, with sharp faces and large ears.

Abnormal cognitive and behavioral abnormalities in women with full mutations are usually milder than those observed in men who develop the disease. Half of them show typical mental retardation and non-verbal learning difficulties, and the other half usually need to be Continuous support for mental retardation, but all of these women seem to have cognitive impairments that originate in the right and frontal lobes, resulting in abnormalities such as vision-space, perceptual skills, executive function, attention and ability to perform simultaneously, however, they Performing well in a continuous process, such as organizational information or chronological order, these women have similar ability in word memory and reading, poor in mathematics, using the Wechsler Children's Intelligence Scale (WISC-R) test In intelligence, their scores in mathematics and graphic arrangement are particularly low, and they also have a special way of speaking, which is repeated and disorganized.

Their personality is often shy and retreat, with strange communication methods and quirks. Special performances include stereotyped behavior, off-topic speech, impulsiveness, distraction and difficulty in adaptation. 20% to 60% of women with full mutations will Being diagnosed with psychosocial disorders, the most common diagnoses are depression, characteristic schizophrenic changes, extensive developmental disorders, personality withdrawal and anxiety.

Pediatric fragile X chromosome examination

Cytological test

(1) Fragile X chromosome examination: The examination of the fragile X chromosome is very important for understanding the frequency of expression of the fragile part and the structure of the chromosome at the fragile part, and is the basic means for confirming the original proband, but the detection rate of the method is low. Generally, it is only about 50%. Because there are other fragile parts (such as FRQXD) that are not related to mental retardation on the X chromosome, even if the presence of the fragile X site is detected, it cannot be diagnosed as a FXS patient or carrier. Therefore, This test can only be used as a screening test and cannot be used as a tool for diagnosis.

(2) Fluorescence in situ hybridization technique: FISH detection can be performed on patients suspected of having a large fragment of the FMR-I gene, and the fluorescently labeled probe is used to treat the chromosomes of the cells in the metaphase division after treatment with colchicine. In situ hybridization showed that the normal chromosomes showed fluorescence, while the chromosomes with corresponding deletions showed no fluorescence.

2. Genetic testing

(1) Southern Blot: Dynamic mutations and deletion mutations of large fragments cause significant changes in the length of the FMR-I gene fragment, so they can be detected by Southern blotting. For different types of mutations of the FMR-I gene, Different restriction enzymes and probes, EcoRI or HindIII restriction enzymes plus pE5.1 (or afaxal) probes can detect pre-mutations of full or large fragments, and are therefore suitable for detecting mental abnormalities. Proband, BclI endonuclease plus StB12.3 probe can detect the diffuse zone containing CGG, can detect and preliminarily determine the size of the pre-mutation, Pst I endonuclease plus OX0.55 The probe can detect the pre-mutation with a small number of CGG repeats. If some restriction enzymes sensitive to methylation, such as EagI, BssHI, SacI, etc., can be used, the methylation of CpG islands can be detected. The whole mutation, pre-mutation and chimeric type are detected. The Southern blotting technique is sensitive and accurate. It is a classical detection method, but the technique is complicated, and it is not suitable for the screening of common populations or high-risk groups, nor can it accurately determine the number of CGG repeats.

(2) Polymerase chain reaction (PCR) technique: PCR amplification of the FMR-I gene fragment of the patient is performed by using appropriate primers, and the amplified product is directly separated by denaturing polyacrylamide gel electrophoresis, and the result can be accurately determined. The number of repetitions of CGG, which is used to find the pre-mutation with a small number of repeats and to observe the (CGG)n distribution in the general population, thereby determining the boundary of the number of CGG repeats between the normal and the pre-mutation. This method is simple and suitable for general screening. The disadvantage is that the full mutation sequence of CGG repeats contains a large number of GC bases, and the PCR reaction is difficult. The PCR technique cannot detect methylation, so the chimeric type cannot be detected.

3. Protein detection

Since FMRP is expressed in almost every tissue and cell in normal humans, but not expressed or abnormally expressed in FXS patients, the presence of this protein can be detected by immunohistochemistry or immunofluorescence using anti-FMRP monoclonal antibody. In the early days, only the blood smears of suspicious people were tested by this method. Recently, fetal exfoliated cells in amniotic fluid were used to observe the presence of FMRP as an indicator for prenatal diagnosis of FXS.

Regular B-ultrasound, electrocardiogram, EEG, etc., can be found in large testicles, abnormal EEG waveforms.

Diagnostic identification of fragile X chromosome in children

diagnosis

The clinical manifestations of the fragile X chromosome are diverse, and the personality, psychological and spiritual changes are not completely the same. Moreover, some patients have clinical symptoms that are not very typical. It is difficult to make a diagnosis based on clinical manifestations. Laboratory tests are not only Early diagnosis ensures a reliable basis, as well as carrier diagnosis and prenatal diagnosis, as well as family surveys and population surveys.

The detection of X fragile sites by cytology is a morphological method, but the accuracy and sensitivity are not very high. Although genetic testing is the main method for diagnosing FXS, genetic testing cannot completely replace chromosome detection because In-depth study found that the fragile X chromosome is not only related to the fragile site FRAXA, but also related to FRAXE, which is considered to be a common fragile site. Recently, it has been found that the FRAXF site also appears to be related to the fragile X chromosome, but currently only related to FRAXA. Mutation of the FMR-I gene, so only genetic testing is easy to miss.

Southern blotting can detect pre-mutation, chimera, full mutation and large fragment deletion, but it is less effective for pre-mutation and deletion of smaller fragments. PCR is suitable for detecting pre-mutation with small number of repeats, but not for detection. Basic.

Differential diagnosis

Different from other hereditary diseases with mental retardation and special face, it mainly relies on laboratory examination for differential diagnosis.

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