A novel RNA-splicing mutation in COL1A1 gene causing osteogenesis imperfecta type I in a Chinese family.

BACKGROUND: Osteogenesis imperfecta (OI), also known as brittle bone disease, is a rare heterogeneous group of inherited disorders characterized by low bone mass and increased bone fragility. The four major clinical criteria for diagnosis of OI are osteoporosis with abnormal fragility of the skeleton, blue sclera, dentinogenesis imperfecta, and premature otosclerosis. The presence of two of these abnormalities confirms the diagnosis. More than 90% patients have autosomal dominant mutations in one of the two genes, COL1A1 and COL1A2, that encode the alpha chains of type I collagen. While the diagnosis of OI is still based on clinical and radiological grounds, there is a growing demand for the molecular characterization of causative mutations. Although there have been several studies on the mutational spectra of COL1A1 and/or COL1A2 in Western populations, very few cases have been reported from Asia. The purpose of this study is to report two patients with OI type I in a Chinese family, who had a novel RNA-splicing mutation in COL1A1 gene and describe the molecular, radiological and clinical findings. METHODS: The proband, (case II-5), a 32-y-old Chinese male, and his 7-y-old daughter were diagnosed as OI type I according to their clinical and radiological features. Genomic DNA was extracted from their blood samples and all promoters, exons and exon/intron boundaries of COL1A1 and COL1A2 genes were sequenced. Polymerase chain reaction sequence-specific primers (PCR-SSP) was used to confirm patients' heterozygous state. RESULTS: Direct DNA sequencing analysis of COL1A1 gene revealed a splicing mutation (c.1875+1G>A, also as IVS 27+1G>A) that converted the 5' end of intron 27 from GT to AT. This mutation was found in both 2 affected individuals but 9 unaffected relatives and the 50 controls were not observed, which was consistent with the clinical diagnosis. This mutation (c.1875+1G>A) appeared to be novel, which is neither reported in literature nor registered in the Database of Collagen Mutations. The heterozygous states of patients' intron 27 were confirmed by PCR-SSP. CONCLUSION: We identify a novel RNA-splicing mutation (c.1875+1G>A) in COL1A1 gene resulting in OI type I in a Chinese family. The detailed molecular and clinical features will be useful for extending the evidence for genetic and phenotypic heterogeneity in OI and exploring the phenotype-genotype correlations in OI.

Rapid molecular prenatal diagnosis of spondyloepiphyseal dysplasia congenita by PCR-SSP assay.

Heterozygous mutations of COL2A1 gene are responsible for type II collagenopathies. The common skeletal phenotypes include achondrogenesis type II, hypochondrogenesis, Stickler dysplasia, Kniest dysplasia, late onset spondyloepiphyseal dysplasia, and spondyloepiphyseal dysplasia congenita (SEDC). Prevention of SEDC can be achieved by prenatal diagnosis. This study reports the first rapid molecular prenatal diagnosis of SEDC performed in China by polymerase chain reaction sequence-specific primer (PCR-SSP) analysis. The pregnant woman we previously reported with SEDC carried the G to A substitution at nucleotide 1510 in exon 23 of COL2A1 gene, which caused a change from glycine to serine at codon 504 (G504S). By the time the woman got pregnant again, she had terminated two pregnancies and still had no child. In the first pregnancy, the molecular mutation of the family was not yet identified, and therefore prenatal diagnosis was unable to be performed by DNA analysis. In the second pregnancy, G504S mutation was found from fetal DNA. At the time of her third pregnancy, the woman and her husband became extremely worried about the potential SEDC for the fetus. For this reason, a quick and reliable molecular prenatal diagnosis of SEDC was performed by a PCR-SSP on an amniocyte sample collected at the 14th week of pregnancy. No mutation of the fetal DNA was identified. The result was obtained within 24 h after the sample was collected. The technique could be applied in confirmatory diagnosis and prenatal diagnosis for the affected family.

[Direct multiplex-PCR from whole blood for rapid detection of Y chromosome microdeletions].

OBJECTIVE: To establish a rapid and simple method to detect Y chromosome microdeletions directly using whole blood as starting material for multiplex-PCR. METHODS: Using a self-prepared pHpH-Bufferq, multiplex-PCR amplification was directly carried out from the anticoagulant whole blood sample without DNA extraction step. Twelve sequence tagged sites (STS), namely SY84, SY86, SY127, SY134, SY124, SY132, SY152, SY157, SY239, SY242, SY254 and SY255, in AZFa, AZFb, and AZFc gene regions were detected in 5 different tubes. In order to ensure the validity of the experiments, sex-determining region Y (SRY) and X-linked or Y-linked zinc finger gene (ZFX/Y) were used as internal controls. Furthermore, conventional PCR using genomic DNA extracted from each blood sample was performed in parallel for evaluating the accuracy of the experiments. RESULTS: A total of 156 male samples were detected, and a normal male sample and a female sample were used as a positive and a negative control respectively. The results showed that 144 samples had no deletion; one sample was AZF-deleted; one sample was AZFb-deleted; seven samples were AZFc-deleted; one sample was both AZFb- and AZFc- deleted; and two samples were all AZFa-, AZFb- and AZFc- deleted. The observed results from two kinds of starting material (whole blood and purified DNA) are completely consistent. CONCLUSION: In our method, PCR amplification was directly carried out from whole blood without any DNA extraction step. So it has the advantages of low cost, simple process, time-saving operation and less cross-contamination. The whole process can be completed within 2 hours. Thus the efficiency of clinical detection is improved greatly.

[A girl with partial monosomy 18q21: cytogenetic and molecular genetics studies].

This study is about a girl with chromosome deletion of 18q and with mental retardation and mild delay of physical development. Based on karyotyping of high resolution, fluorescence in situ hybridization (FISH) and microsatellite analysis mapping to 18q, we found that the patient's karyotype was interpreted as 46,XX,del(18).(pter-->q21:), ish del(18)(D18Z1+,qter-). Detection of D18S979 showed that the region from 18q21.1 to 18qter was deleted, which was originated from her father. There were MBP gene and GALNR gene in the deleted interval in which both of them were lost. In conclusion, deletion of 18q21-->qter including the MBP gene and GALNR gene should be responsible for her mental retardation and mild delay of development.

[AZF microdeletions are not related with recurrent spontaneous abortion].

OBJECTIVE: To study the relationship between azoospermia factor (AZF) microdeletions of the Y-chromosome and recurrent spontaneous abortion. METHODS: We collected 26 chorionic villus samples from abortive male embryos and 51 blood samples from the husbands whose wives had recurrent spontaneous abortion, extracted genomic DNA from the samples and detected 12 STSs in the AZFa, AZFb and AZFc regions of Yq11.2 by amplification multiplex PCR. RESULT: AZF microdeletions were found neither in the chorionic villus samples nor in the blood samples. CONCLUSION: There is no relationship between AZF microdeletions and recurrent spontaneous abortion.

[Evaluation of CD4+ CD25+ regulatory T cells in the peripheral blood of recurrent spontaneous abortion patients].

OBJECTIVE: To explore the role of CD4+ CD25+ regulatory T cells (CD4+ CD25+ Tr) in the pathogenesis of recurrent spontaneous abortion. METHODS: Peripheral blood samples were collected from 29 women with unexplainable recurrent spontaneous abortion (the URSA group) and another 20 with normal pregnancy (the control group). The percentage of CD4+ CD25+ Tr in the peripheral blood was measured by flow cytometry. RESULTS: The rate of CD4+ CD25bright Tr in the URSA patients ([1.98 +/- 0.96]%) was significantly lower than that in the control group ([3.21 +/- 1.25]%, P < 0.05), while the percentages of CD4+ CD25+ and CD4+ CD25dim and the ratio of CD4+ CD25bright/CD4+ were not significantly different between the two groups (P > 0.05). CONCLUSION: URSA might be associated with the decreased percentage of CD4+ CD25bright Tr, which plays an important role in fetomaternal immunologic tolerance.

[Observation of spermatogenic cells for infertile patients with Y-chromosomal microdeletion].

OBJECTIVE: To assess the spermatogenic function of the infertile patients with Y-chromosomal microdeletion. METHODS: Thirty-five 23-44 years old patients with microdeletions of Y chromosome were included in this study. Three semen analyses confirmed that 26 cases were non-obstructive azoospermia and 9 oligospermia with sperm count < 1 x 10(6)/ml. They were divided into 3 groups by the locus of deletion, 5 cases of AZFa + b + c deletion in group 1, 4 cases of AZFb + c and 3 cases of AZFb deletion in group 2, and 23 cases of AZFc deletion in group 3. Semen was collected and centrifuged, the supernatant removed and the centrifugate applied on the clean slides after dilution. Following Wright's-Giemsa staining, the slides were viewed under the microscope. Testis histopathological biopsy was performed for 6 of the cases. RESULTS: In group 1, no spermatogenic cells were observed but only Sertoli cells in 1 case, with a consistency between the result of spermatogenic cell test and that of testis biopsy. In group 2, spermatogenic cell tests revealed spermatocytes in 6 cases, 2 were proved by testis biopsy with sperm maturation arrest in the primary spermatocyte stage, and spermatogenic cells of all developmental stages were seen in 1 AZFb deletion patient with the same sperm maturation arrest as the above two. In group 3, only primary spermatocytes were detected by spermatogenic cell test in 5 oligospermia patients but no spermatogenic cells in the 15 azoospermia cases, and biopsy revealed 2 cases of Sertoli cell-only syndrome. CONCLUSION: The spermatogenic cell test can effectively assess the spermatogenic function of AZF deletion patients. Non-invasive and easily accepted by patients, it is highly recommendable for the evaluation of spermatogenesis of patients with Y-chromosomal microdeletion.

[Prenatal diagnosis of two pregnancies with risk of chromosomal disorders].

OBJECTIVE: To report the prenatal diagnosis of 2 cases of pregnancy with the risk of chromosomal disorders. In Case 1, the pregnant woman had a daughter with testicular regression syndrome and a segmental duplication of Ypter --> Yp11.2 and a deletion of Yq11.23 --> Yqter. In Case 2, both the pregnant woman and her husband were carriers of chromosomal balanced translocation. METHODS: Two samples of amniotic fluid were obtained at the 19th week of gestation for fetal karyotype analysis. For Case 1, FISH with a probe of Xp/Yp subtelomere was performed on the metaphase of the amniotic fluid, genomic DNA of the amniotic fluid extracted and multiplex PCR conducted for AZF regions. Both the pregnant women underwent sonography to confirm the karyotypic diagnosis. RESULTS: Cytogenetic, FISH and multiplex PCR analysis of the cultured amniotic fluid cells from Case 1 showed a normal male karyotype, and ultrasound scan of the fetus showed normal male external genitalia and normal development. Cytogenetic analysis of the cultured amniotic fluid cells from Case 2 revealed a karyotype of balanced translocation with t(13 ; 14) from the father, and no abnormality of the fetus was found by ultrasound scan. CONCLUSION: It is helpful to perform cytogenetical and molecular prenatal diagnosis in combination with ultrasound scan for the fetus with the risk of chromosomal disorders and subsequently for genetic counseling.

[Clinical, molecular and cytogenetic studies on 4 patients with 46, XX (SRY positive) male syndrome].

OBJECTIVE: To analyze the clinical, molecular and cytogenetic features of 46, XX (SRY positive) male syndrome. METHODS: The clinical features of 4 patients with 46, XX (SRY positive) male syndrome were analyzed retrospectively. Karyotyping, FISH, PCR amplification of the SRY gene, and Y-chromosome microdeletion were performed to study their molecular cytogenetic features. RESULTS: The Four patients were all sociopsychologically males of short stature and came to hospital for infertility. Physical examination revealed that their testes were small in volume and soft in texture, but their penes were normal. Semen analyses showed complete azoospermia. Detection of serum sexual hormone suggested hypergonadotropic hypogonadism. All were karyotyped as 46, XX. Molecular analyses revealed the presence of the SRY gene and absence of AZFa, b and c of the Y chromosome. FISH analysis showed that SRY genes were translocated to Xp in 3 of the patients. CONCLUSION: Phenotypically 46, XX (SRY positive) male patients are males generally, for the presence of the SRY gene in the whole genome and azoospermia due to the deletion of AZF. The clinical characteristics of the patient include testis dysgenesis, infertility and short stature. The long arm of the Y chromosome might contain the gene associated with body height. Extensive molecular and cytogenetic studies on 46, XX male syndrome may help to elucidate its genotype-phenotype relation.

A case of agonadism associated with y-chromosome rearrangement: cytogenetic and molecular studies.

Testicular regression syndrome (MIM273250) is characterized primarily by absence of gonads in a person of XY karyotype. Phenotypes range from complete female external genitalia (primary or "true" agonadism) to male phenotype with anorchia (testicular regression). Phenotypic differences depend on the stage of embryo development during which testes degenerate. No conclusive mapping can be concluded for the phenotype. We describe a novel case of primary agonadism with a karyotype of 46,X,der(Y)(pter-->q11.23::pter-->p11.31 or p11.2:). Transcriptional analysis revealed little expression of USP9Y and UTY genes on the Y chromosome in our case, which would explain her phenotypes of agonadism with short stature.