Effects of antibiotics on motility, sperm morphology, membrane integrity, fertility and bacteriological quality of buffalo spermatozoa

Abstract

Over the past few years several different types of Human Skin Disorders have been characterized at clinical and molecular leve ls. In a number of cases of skin di sorders, not only chromosomal localizations but aberrations in the genes invo lved have been identified. The discovery of the disease genes has revolut ion ized understanding about better diagnosis and therapeutics of hered itary skin abnormalities. Pakistan, due to its ethnic d iversity and social customs, harbor large consanguineous fam ilies, which are the major source of traditional methods for mapping disease genes. For the study, presented in my PhD dissertati on, I travelled to some of the very remote areas of Pakistan and studied eighteen fam ilies with skin disorders. Travelling to some of these areas is one of the most exhaust ive and weari some experience of my life. Nonava ilability of hospita ls, proper transport facilities and even roads in some ofthe areas has made the experience more remarkable and unforgetta ble. In the present study, six fam ilies with hered itary ectodermal dysplasias (A-F) and twelve fa milies with hereditary ha ir loss (G-R) have been characterized both at clinical and molecular levels. Affected indi viduals in each fam ily were thoroughly examined for a ll kinds of abnormalities especia lly those affectin g skin, ha ir, na il, teeth and sweat glands. Each family was then characterized at molecular level by testing linkage to the known/unknown genes and DNA sequencing using automated DNA sequencer. ]n a couple of fami lies, human genome was scanned using more than 500 highly polymorphic microsate llite markers to locate the disease gene. In fa mily A, affected individua ls presented features of hypohidrotic ectodermal dysplasia segregating in an autosomal recessive manner. The two affected individua ls in the fami ly showed c linical features including fine and sparse scalp hair, absent eyebrows and eyelashes, absent axillary and pubic hair, conical teeth, diminished sweating, dry and thin skin, protruding prominent lips, pointed chin, fronta l boss ing, hyperpigmentation of the skin around the eyes and mouth, and saddle-shaped nose. Genotyping data estab lished linkage in the family to gene EDAR on chromosome 2q ll -q 13. Subsequently, seq uence ana lys is of the gene revea led a homozygous sp lice donor s ite mutation (c.442+ 1 G>C, IVS5+ 1 G>C) in affected individuals of the fami ly. ]n family B, fourteen individuals showed typ ical features of X-linked recessive ichthyosis (RXLl). Analys is of family pedigree and c linical feat ures of the affected indi vidua ls led directly to screening of the gene STS, mapped earl ier on chromosome Xp22.32. Analysis of the PCR results and DNA seq uencing detected a complex double de letion in DNA of the affected indiv idua ls. Thi s invo lved de letion of exons 1-2 and 5-10, and > than 1.2 Kb upstream and downstream DNA seq uence of the gene STS. In family C, al l three affected ind ivid ua ls exh ibited features of lipo id proteinosis includi ng warty ski n, diffuse acneiform scars on the ski n, infiltration on the face and hands, and ye llow erythocytic lesions on the hands. Genotyp ing resu lts showed linkage of the fam ily to gene ECMI, located on chromosome I q2 1. However, sequence analysis fa iled to identify potential sequence variants in the gene ECMI in DNA of the affected individuals. Six individuals in two fam ilies (0 and E), prese nted characteristic features of a condition of miss ing fin gerna ils and toena ils called anonychia congenita. Linkage in both the fa milies was establi shed to a recently di scovered gene RSP04, mapped on chromosome 20pl 3. Sequence ana lysis fa iled to detect any potential seq uence variant in the gene RSP04 in affected ind ividuals of the fa mily O. However, in the other fa mily (E), sequence analys is revealed a novel nonsense mutat ion (c. 18C>A; p.Cys6X) in the only affected individua l. In fa mily F, a ll three affected individua ls showed features representing a novel form of ectoderma l dysplas ia. Affected individuals exhibited features inc luding sparse scalp ha ir, sparse eyebrows and eyelashes, sparse beard and moustache in male affected individuals, and dystrophic nails. Skin in the affected indi vidua ls was thin and not eas ily healed on receivi ng the inj ury. Hyperh idros is was observed on palms and so les of the affected members. Human genome scan using more than 500 polymorphic microsate llite markers, established linkage in the fa mily to chromosome 20pl3. The highest two-po int LOO score, at zero recombination fraction (8=0.00), of 1.97 was obta ined at a marker 0 20S482. Maximum multi-point LOO score of 2.65 was achieved at three markers (020S 199, 020S 11 3, 020S842) a long the disease-i nterval. Sequenci ng of two cand idate genes (RSP04, TGM3), located in the linkage interval of 3.88 Mb, in two affected and one unaffected member of the fa mily fa iled to detect potential sequence variants. Of the twelve families with hereditary ha ir loss di sorders, presented here, the fa mily G showed segregati on of woolly ha ir in a utosomal dominant fas hion. Affected individuals exhibited short tightl y curled twisted ha ir entangled with each other. Linkage in the fa mily was estab li shed to type II keratin gene KRT74 on chromosome l 2q 12-1 4.1. Sequence ana lysis of the gene KRT74 identified a novel splice site mutation (c.lVS8- I G>A) in all nine affected individua ls of the fa mily. In s ix families (H, I, J, K, L, M), affected individuals presented features of hered itary hypotrichosis. The affected ind ividua ls showed absent/sparse/wooly sca lp hair, sparse to absent eyebrows and eyelashes, and missing axillary and pubic hair. In family H linkage was establ ished to DSG4 gene on chromosome] 8q1 2.l-q1 2.2, in two families (I, J) to LlPH gene on chromosome 3q27 and three other families (K, L, M) to the gene LPAR6 on chromosome 13q 14.] l-q23 .21. Sequence analysis of the gene DSG4 detected a previously reported deletion mutation (Ex5 _ 8del) in all the affected individuals of the family H. In family I and J, screening of the gene LIPH revealed 2 base pairs homozygous deletion mutation (c.659 660delT A) leading to frameshift and premature termination codon 69 base pairs downstream of the mutation point (p.Ile220ArgfsX25). Screen ing of the gene LP AR6 detected a previously repotied 4-bp insertion mutation (c.69insCATG; p.24insHisfsX52) in affected individuals of families K and L and a missense mutation (c.562A>T; p.Ile188Phe) in affected members offamily M. In three families (N, 0 , P), affected individuals showed typical features of atrichia with papular lesions (APL). All eighteen affected individuals in the three families showed complete absence of scalp hair, eyebrows, eyelashes and other body hair. Linkage in the families was established to gene HR on chromosome 8p21.3 . Sequence analysis revealed a novel nonsense mutation (c.2458C>T; p.Arg819X) in exon 11 of the gene in family N. In the other two families (0 and P), sequence analysis failed to identify potential sequence variants in the gene HR. In two families (Q and R), a novel form of hair loss was observed. In family Q, affected individuals presented features of late onset of hair loss, which ultimately lead to complete hair loss from all body parts including scalp, eyebrows, eyelashes and pubic. Linkage in the family was tested with several other hair loss genes, but no evidence of linkage was found. In family R, twelve individuals showed late onset of hair loss. In all the affected individuals, loss of hair started from top of the scalp at the age of 25 years, which ultimately led to complete baldness at the age of 30-35 years. Human genome scan using more than 500 microsatellite markers failed to detect linkage in the family on any chromosome. The data obtained from the present study contributed in publishing the following articles 1. Wasif N, Naqvi SK, Basit S, Ali N, Ansar M, Ahmad W (2011) Novel mutations in the keratin-74 (KRT74) gene underlie autosomal dominant woolly hair/hypotrichosis in Pakistani families. Human Genetics 129: 419-424 2 . Azeem Z, Wasif N, Basit S, Sohail M, Ahmad W (2011) HR mutations underlies Atrichia with Papular Lesions in four Autosomal Recessive Pakistani Families.