Molecular characterization of de novo ring chromosome 21 in a child with seizures, growth retardation, and multiple congenital anomalies.

The ring chromosome 21[r(21)] syndrome is a rare disorder, and mainly occurs as a de novo event. However, a wide variation of the phenotype has been reported in r(21) cases depending on breakpoints, loss of genetic material, and mosaicism of cells with r(21) and monosomy 21, causing copy number alterations. A 29-month-old female was referred to the centre for seizures, developmental delay, microcephaly, hypotonia, deafness, and other congenital abnormalities. Physical examination revealed short stature and multiple facial dysmorphism. She was unable to sit, walk or stand by herself. Cytogenetic study with GTG banding revealed a karyotype of mos 46,XX,r(21)(p11.1q22.12)[70]/45,XX,-21[10]/47,XX,r(21),+r(21)[1]/46,XX[10]. Additionally, molecular cytogenetics refined the breakpoints and characterized the deleted region (RP11-410P24/CHR21: 32849565-33019511) in the clone with the r(21) as ~12-14 Mb contiguous region at 21q22.12 to 21qter. The present study has accurately detected copy number alterations caused by ring chromosome formation. The basis of the UCSC Genome Browser on Human (GRCh38/hg38) analysis suggests hemizygous expression of a deleted critical region of chromosome 21 in ring chromosome cell lines. This is likely to be the underlying cause of the present phenotypes in the patient. Overall, the genotype-phenotypic correlation in r(21) cases remains widely diverse, most likely due to tissue-specific mosaicism of the 45, XX,-21 cell line.

The association of testis-specific hTAF7L gene variants with idiopathic azoospermic and severe oligozoospermic male infertility.

Spermatogenesis is regulated by complex tissue specific gene expression in the testis to achieve normal male fertility. X-chromosome specific TATA binding protein (TBP)-associated factor 7L (hTAF7L) is one of the transcriptional regulator genes considered essential for spermatogenesis. The aim of this study was to evaluate the role of variants/mutations in the testis-specific hTAF7L gene in non-obstructive azoospermia and severe oligozoospermia male infertility. We studied 156 idiopathic non-obstructive azoospermic, severe oligozoospermic infertile males and 50 fertile proven controls. Infertile males and control subjects were genotyped for variants of the hTAF7L gene using polymerase chain reaction and a direct Sanger sequencing approach. The odds ratio evaluated the association of hTAF7L gene variants with idiopathic male infertility. The variants found in the hTAF7L gene were subjected to an in-silico analysis study. In infertile study subjects, we observed 11 single base pair nucleotide changes at various exons and three frameshift variants at exon 10 in the hTAF7L gene. We also found more than one variant in some non-obstructive azoospermia and severe oligozoospermia infertile males along with control subjects. All these variants changed the amino acid sequences in the hTAF7L gene. However, similar changes were also seen in fertile subjects, and the differences were not statistically significant. In-silico tools also predicted that the variants were likely to be benign. The variants in cDNA of the hTAF7L gene were typical SNPs. It is found that the hTAF7L gene is highly polymorphic and these missense variants are not directly associated with male infertility. However, we feel that more studies are needed to elucidate the role of multiple variants of the hTAF7L gene in the process of normal spermatogenesis.

A missense mutation (c.226C>A) in HMG box SRY gene affects nNLS function in 46,XY sex reversal female.

The SRY initiates cascade of gene expression that transforms the undifferentiated gonad, genital ridge into testis. Mutations of the SRY gene is associated with complete gonadal dysgenesis in females with 46,XY karyotype. Primary amenorrhea is one of the clinical findings to express the genetic cause in 46,XY sex reversal. Here, we report a 26-year-old married woman presenting with primary amenorhea and complete gonadal dysgenesis. The clinical phenotypes were hypoplastic uterus with streak gonad and underdeveloped secondary sexual characters. The cytogenetic analysis confirmed 46,XY sex reversal karyotype of a female. Using molecular approach, we screened open reading frame of the SRY gene by PCR and targeted DNA Sanger sequencing. The patient was confirmed with nucleotide substitution (c.226C>A; p.Arg76Ser) at in HMG box domain of SRY gene that causes 46,XY sex reversal female. Mutation prediction algorithms suggest that alteration might be disease causing mutation and mutated (p.Arg76Ser) amino acid deleteriously affects HMG box nNLS region of SRY protein. Clinical phenotypes and in silico analysis confirmed that missense substitution (p.Arg76Ser) impaired nNLS binding Calmodulin-mediated nuclear transport of SRY from cytoplasm to nucleus. The mutation affects down regulation of male sex differentiation pathway and is responsible for 46,XY sex reversal female with gonadal dysgenesis.

Assessment of Oxidative Stress Biomarkers and Body Mass Index in Pulmonary Tuberculosis Patients: A Case-Control Study.

Background: Pulmonary tuberculosis (PTB) is a major public health concern in most underdeveloped and developing countries. PTB affects the nutritional status of the patients and influences the body mass index (BMI). There is tissue inflammation and free radical burst from activated phagocytes resulting in oxidative stress. The present study was designed to assess the relationship between oxidative stress and body mass index in newly detected pulmonary tuberculosis patients. Method: This was a case-control study designed to assess oxidative stress parameters such as nitric oxide (NO) and malondialdehyde (MDA) in 40 consecutives newly diagnosed PTB patients and compared with 40 age-matched healthy controls. The nutritional status of the study subjects was measured by calculating the BMI. Results: The mean BMI was 21.61±3.52 Kg/m2 in controls and 17.47±1.56 Kg/m2 in PTB patients and the difference was statistically significant (p <0.0001). The mean levels of MDA (7.65±0.65 nmol/ml) and NO (36.12±1.07 µmol/l) were significantly higher in PTB patients compared to controls (MDA 3.56±0.41 nmol/ml and NO 14.48±0.93 µmol/l). Conclusions: Increased levels of malondialdehyde and nitric oxide were observed in newly diagnosed PTB patients when compared to controls indicating oxidative stress in PTB. The BMI of these patients was significantly lower than the controls. Thus, it is concluded that there is an inverse relationship between oxidative stress and BMI in PTB patients and antioxidant supplementation in addition to nutritional intervention under the National Tuberculosis Elimination Program may help to improve the BMI and promote better recovery in these patients.

Oxidative stress and its impact on mitochondrial DNA in pulmonary tuberculosis patients- a pilot study.

BACKGROUND: Pulmonary tuberculosis (PTB) remains a major cause of morbidity and mortality all around the world. Recent studies have pointed out increased oxidative stress and also DNA damage in peripheral blood in PTB. Till date, to the best of our knowledge, no study has so far been conducted to show the mitochondrial DNA (mtDNA) deletions mapping in PTB patients. Therefore we performed the present study with the aim to investigate oxidative stress parameters along with mtDNA damage in newly diagnosed untreated PTB patients. MATERIAL AND METHODS: This is a prospective study carried out in Mahatma Gandhi Institute of Medical Sciences, Sevagram,Wardha, Maharashtra during september 2017 to september 2018.Thirty newly diagnosed untreated PTB patients and thirty age matched healthy controls were enrolled in the present study. Analysis of Oxidative stress parameters such as nitric oxide (NO) and malondialdehyde (MDA) were done by calorimetric methods. Assessment of mitochondrial DNA damage was carried out by mtDNA deletions mapping using primer shift long range polymerase chain reaction technique. RESULTS: There was significant increase in levels of oxidative stress parameters, nitric oxide and malondialdehyde, in PTB patients compared to controls (p < 0.01). Generally there are two common deletion sites of "13 bp direct repeats" (ACCTCCCTCACCA) in mtDNA. One at the junction sites from bp 8470 to 8482 bp and another from bp 13447 to 13460 bp which make mtDNA more prone for 4977bp deletion. Out of thirty cases of PTB, two cases showed mtDNA damage in the form of mtDNA deletion of 4977bp. There was no mtDNA deletion in any control which can be attributed to continuous generation of oxidative stress. CONCLUSION: This pilot study has been able to demonstrate that compared to controls, in newly diagnosed pulmonary tuberculosis patients some mtDNA damage did occur and was probably due to continuous generation of oxidative stress in tuberculous patients. However, sample size is too small to draw any conclusions but definitely a more comprehensive study, by recruiting more number of pulmonary tuberculosis patients is warranted to establish correlation between oxidative stress and mtDNA damage in PTB.

A Study on Chromosomal Analysis of Patients with Primary Amenorrhea.

BACKGROUND: Primary amenorrhea is one of the most common disorders seen as gynecological problems in adolescent girls. It refers to the participants who did not attain menarche by the age of 11-15 years. Chromosome abnormalities contribute as one of the etiological factors in patients with primary amenorrhea. AIMS: The aim of this study was to evaluate the frequency of chromosomal abnormalities and to investigate the abnormal karyotypes in patients referred with the symptom of primary amenorrhea for better management and counseling. SETTING AND DESIGN: One hundred and seventy-four cases of primary amenorrhea were referred from the obstetrics and gynecology department to our cytogenetic laboratory for chromosomal analysis. G-banded chromosomes were karyotyped, and chromosomal analysis of all patients was done. RESULTS: Out of 174 patients, we observed 23 (13.22%) participants with abnormal karyotype. In 23 cases of chromosomal abnormalities, 10 cases were sex reversal female (46,XY) and Turner karyotype (45,X) in 6 females. Other numerical and structural abnormalities were also seen such as 47,XXX; 45,X/47,XXX; 45,X/46, X,dic(X); 46,XX, inv (9); 45,X/46,X,i(Xq); 46,X,mar(X); and 45,X/46,XY in the primary amenorrhea cases. CONCLUSION: This study definitely attests the importance of chromosomal analysis in the etiologic diagnosis of primary amenorrhea patients. Karyotyping will help to counsel and manage the cases of primary amenorrhea in a better way. This study reveals the frequencies and different types of chromosomal abnormalities found in primary amenorrhea individuals and that might help to make the national database on primary amenorrhea in relation to chromosomal aberrations.

Molecular Genetic Study to Detect Prevalence of High-risk Human Papilloma Virus Strains (type 16 and 18) in Cervical Lesions and Asymptomatic Healthy Subjects of Rural Central India.

BACKGROUND: Carcinoma cervix of uterus (CaCx) is the most common malignancy affecting women worldwide. It is an established fact that infection of specific types of human papilloma virus (HPV) is essential for the development of cervical cancer. The present study reports the high-risk viruses (HPV 16 and 18) type distribution in rural central India, which has unique climatic condition. To our knowledge, no molecular study on HPV prevalence has been done in this region of rural population, this intended us do such study. MATERIALS AND METHODS: Sexually active women reporting to the Gynecology were divided in three groups, first being asymptomatic women with normal cervix (52 cases), second group with benign cervical lesion (52 cases), and third group of women with frank cervical malignancy (40 cases). Cervical swabs were collected for HPV DNA sampling. The incidence of HPV positivity was recorded in each group. RESULTS: Fifty-two women with asymptomatic normal cervix showed 44.23% positivity for HPV 16 and 5.76% positivity for HPV 18. Fifty-two women with benign cervical lesion showed 38.46% positivity for HPV 16 and 3.84% positivity for HPV 18. Forty women with frank cervical malignancy were with prevalence of 62.5% for HPV 16 and 22.5% for HPV 18. CONCLUSION: The results of the study are definitely helpful to know the prevalence of HPV in this region of rural population and will enrich the national epidemiological data related to HPV infection in cervical cancer.

Chromosomal Aberrations in Couples with Pregnancy Loss: A Retrospective Study.

BACKGROUND: Recurrent pregnancy loss is a challenging reproductive problem, and chromosomal anomalies approximately affect 2%-8% of couples with recurrent pregnancy loss. The chromosomal abnormality, especially balanced translocation rearrangement in either parent, is the important cause of recurrent spontaneous abortion. AIMS: The aim of this study was to investigate the role and prevalence of chromosomal anomalies in recurrent miscarriages. The results will be helpful for counseling and make the decision for alternative options and precaution for the affected couples and also support to make a national database. SETTINGS AND DESIGN: The present retrospective study was carried out in 172 couples (344 individuals) having the history of three or more recurrent spontaneous abortion. The cytogenetic analysis was done in all 344 individuals using G-banding and karyotyping. RESULTS: Out of 172 couples, 17 couples (9.88%) had different types of structural or numerical chromosomal abnormalities. The structural aberrations were observed in 15 (8.72%) couples, and numerical aberrations were seen in 2 (1.16%) couples. Out of 17 couples, 8 (47.05%) had balanced translocations, 2 (11.76%) had the Robertsonian translocation, 5 (29.41%) had the pericentric inversion of chromosome 8, 9, and Y, and only 2 (11.76%) women showed sex chromosome numerical aberrations. CONCLUSIONS: Cytogenetic analysis should be an important routine investigation in couples with repeated miscarriages. Cytogenetic analysis is essential and helpful for genetic counseling to take precaution and implementing proper reproductive alternatives. Studies on the genetic basis of pregnancy loss should be taken up to generate data on these issues from different regions.

Association of large scale 4977-bp "common" deletions in sperm mitochondrial DNA with asthenozoospermia and oligoasthenoteratozoospermia.

OBJECTIVE: To determine the association of large-scale mitochondrial DNA (mtDNA) deletions with abnormal sperm or abnormal flagellar movement of human spermatozoa in asthenozoospermia and oligoasthenoteratozoospermia (OAT) subjects using percoll gradients fractionation and long-range polymerase chain reaction (PCR). DESIGN: We investigated sixty infertile men and thirty normal healthy fertile controls. Of sixty infertile men, 39 were asthenozoospermia and 21 were OAT. MATERIALS AND METHODS: Percoll gradients discontinuous technique was used for separation of spermatozoa on the basis of their motility. Long-range PCR was used for detection of "common" 4977-bp deletions, and primer shift technique was used for confirmation of deletions. RESULTS: Overall fourteen subjects (14/60; 23.3%) of which eight (8/39; 20.5%) asthenozoospermia and six (6/21; 28.6%) OAT had shown deletions of 4977-bp. Deletions were more common (23.3%) in 40% fraction than 60% (11.6%) and 80% (5%) fractions. Sequencing results had shown deleted region of mtDNA. CONCLUSION: Abnormal spermatozoa had more number of mtDNA deletions than normal sperm, and abnormal spermatozoa had lost genes for the oxidative phosphorylation. Our findings suggest that large-scale 4977-bp mtDNA deletions in the spermatozoa from the infertile subjects cause the asthenozoospermic and OAT pathophysiological conditions in infertile males.

Large Scale 7436-bp Deletions in Human Sperm Mitochondrial DNA with Spermatozoa Dysfunction and Male Infertility.

INTRODUCTION: Mitochondria and mitochondrial DNA are essential to sperm motility and fertility. It controls growth, development and differentiation through oxidation energy supply. Mitochondrial (mtDNA) deletions or mutation are frequently attributed to defects of sperm motility and finally these deletions lead to sperm dysfunction and causes infertility in male. AIM: To investigate the correlation between large scale 7436-bp deletions in sperm mtDNA and non-motility of sperm in asthenozoospermia and Oligoasthenoteratozoospermia (OAT) infertile men. MATERIALS AND METHODS: The present prospective study was carried out in Human Genetic Division, Department of Anatomy, Mahatma Gandhi Institute of Medical Sciences, Sevagram from June 2014 to July 2016. We have studied 110 asthenozoospermia and OAT infertile men whose semen profile indicated abnormal motility and 50 normal fertile controls. Of 110 infertile men, 70 had asthenozoospermia and 40 had OAT. Fractionations of spermatozoa were done in each semen sample on the basis of their motility by percoll gradients discontinuous technique. Long-range PCR was used for detection of 7436-bp deletions in sperm mtDNA and was confirmed by primer shift technique. RESULTS: Overall eight subjects (8/110; 7.2%) of which six (6/70; 8.57%) asthenozoospermia and two (2/40; 5%) OAT had shown deletions of 7436-bp. In 40% percoll fraction had more non-motile spermatozoa than 80% percoll fraction. The non-motile spermatozoa in 40% percoll fractions showed more mtDNA deletions (7.2%) than the motile spermatozoa in 80% percoll fraction (2.7%). The sequencing of flanking regions of deleted mtDNA confirmed 7436-bp deletions. Interestingly, no deletions were found in control subjects. CONCLUSION: Though, the frequency of 7436-bp deletions in sperm mtDNA was low in infertile cases but meaningful indications were there when results were compared with controls. It is indicated that large scale deletions 7436-bp of mtDNA is associated with abnormal sperm motility. The 7436-bp deletions of mtDNA in spermatozoa may be one of the important causes of dysfunction and non-motile sperm.

Genetic Risk of Azoospermia Factor (AZF) Microdeletions in Idiopathic Cases of Azoospermia and Oligozoospermia in Central Indian Population.

BACKGROUND: Genetic factors cause about 15% of male infertility. Azoospermia factors (AZFa, AZFb, and AZFc) present on Yq are most important for spermatogenesis. We have made an attempt to evaluate the frequencies of microdeletions of AZFa, AZFb, AZFc in idiopathic cases of azoospermia and oligozoospermia from central Indian population. MATERIALS AND METHODS: We have analyzed a total of 156 subjects (95 oligozoospermia and 61 azoospermia) & 50 control subjects. DNA samples were analyzed for microdeletions of Y chromosome by PCR-screening of 18 sequences-tagged-site (STS) markers from different region of the AZF on Yq and SRY on Yp. RESULTS: Out of 156 cases analyzed, 13 (8.33%) subjects (8 azoospermia and 5 oligozoospermia) showed partial deletion of AZF regions, of which deletion in AZFc region was the most common (84.6%) followed by AZFb (15.4%) and AZFa (15.4%). The sites and sizes of deletions varied among patients. Histological study of the testicular tissue of the available subjects, who showed microdeletions of Y chromosome, showed spermatogenic arrest at different stages. The frequency of Y chromosome microdeletion in our subjects was 8.33%. CONCLUSION: Some Indian studies reported low frequencies of microdeletions than that of our result. We suggest that the frequency of deletions may be affected by the involvement of different genetic factors, ethnic population and different geographical regions. PCR based Y chromosome screening for microdeletions will be useful and great help to infertility clinics for genetic counselling and assisted reproduction.