RDH12-associated retinal degeneration caused by a homozygous pathogenic variant of 146C>T and literature review

AIM:To describe the clinical,electrophysiological,and genetic features of an unusual case with an RDH12 homozygous pathogenic variant and reviewed the characteristics of the patients reported with the same variant.METHODS:The patient underwent a complete ophthalmologic examination including best-corrected visual acuity,anterior segment and dilated fundus,visual field,spectral-domain optical coherence tomography(OCT)and electroretinogram(ERG).The retinal disease panel genes were sequenced through chip capture high-throughput sequencing and Sanger sequencing was used to confirm the result.Then we reviewed the characteristics of the patients reported with the same variant.RESULTS:A 30-year male presented with severe early retinal degeneration who complained night blindness,decreased visual acuity,vitreous floaters and amaurosis fugax.The best corrected vision was 0.04 OD and 0.12 OS,respectively.The fundus photo and OCT showed bilateral macular atrophy but larger areas of macular atrophy in the left eye.Autofluorescence shows bilateral symmetrical hypo-autofluorescence.ERG revealed that the amplitudes of a-and b-wave were severely decreased.Multifocal ERG showed decreased amplitudes in the local macular area.A homozygous missense variant c.146C>T(chr14:68191267)was found.The clinical characteristics of a total of 13 patients reported with the same pathologic variant varied.CONCLUSION:An unusual patient with a homozygous pathogenic variant in the c.146C>T of RDH12 which causes late-onset and asymmetric retinal degeneration are reported.The clinical manifestations of the patient with multimodal retinal imaging and functional examinations have enriched our understanding of this disease.

Development of homozygous tetraploid potato and whole genome doubling-induced the enrichment of H3K27ac and potentially enhanced resistance to cold-induced sweetening in tubers

Polyploid plants typically display advantages on some agronomically important traits over their diploid counterparts.Extensive studies have shown genetic,transcriptomic,and epigenetic dynamics upon polyploidization in multiple plant species.However,few studies have unveiled those alternations imposed only by ploidy level,without any interference from heterozygosity.Cultivated potato is highly heterozygous.Thus,in this study,we developed two homozygous autotetraploid lines and one homozygous diploid line in parallel from a homozygous diploid potato.We confirmed their ploidy levels using chloroplast counting and karyotyping.Oligo-FISH and genome re-sequencing validated that these potato lines are nearly homozygous.We investigated variations in phenotypes,transcription,and histone modifications between two ploidies.Both autotetraploid lines produced larger but fewer tubers than the diploid line.Interestingly,each autotetraploid line displayed ploidy-related differential expression for various genes.We also discovered a genomewide enrichment of H3K27ac in genic regions upon whole-genome doubling(WGD).However,such enrichment was not associated with the differential gene expression between two ploidies.The tetraploid lines may exhibit better resistance to cold-induced sweetening(CIS)than the diploid line in tubers,potentially regulated through the expression of CIS-related key genes,which seems to be associated with the levels of H3K4me3 in cold-stored tubers.These findings will help to understand the impacts of autotetraploidization on dynamics of phenotypes,transcription,and histone modifications,as well as on CIS-related genes in response to cold storage.

Iron Metabolism Abnormalities in Children with Homozygous Sickle Cell Disease in Brazzaville

Introduction: Sickle cell disease is the most common genetic disease in the world, particularly in sub-Saharan Africa. It is a protean condition with multiple complications including disturbed iron metabolism. Objectives: To determine the prevalence of iron metabolism abnormalities in children with homozygous sickle cell disease, to describe the epidemiological, clinical and paraclinical characteristics of children with these abnormalities and to identify associated factors. Patients and Methods: This was a cross-sectional analytical study conducted over 9 months in the mother-child consultation unit of the Brazzaville University Hospital, the National Reference Centre for Sickle Cell Disease and the paediatric department of the Blanche Gomes mother-child hospital. It concerned children aged between 3 months and 15 years followed up for homozygous sickle cell disease. The study was based on a haemogram, iron metabolism test, LDH, transaminases and CRP. Results: The overall prevalence of iron metabolism abnomalities was 40.7%. Of the 145 children included, 35.9% had iron overload and 4.8% iron deficiency. Iron overload was associated with infections, undernutrition, iron supplementation and number of blood transfusions. Iron deficiency was not significantly associated with any factor but recurrent infections were relatively more frequent. Conclusion: Abnormalities of iron metabolism in sickle cell patients are relatively frequent, which justifies monitoring during follow-up for early detection and better management. .

Correlation between Pubertal Delay in Adolescents with Homozygous Sickle Cell Disease and Socio-Demographic, Clinical Factors

Introduction: Pubertal development is a process leading to the acquisition of reproductive capacities. Among the factors that inhibit pubertal development are chronic diseases including sickle cell anemia, which is a public health problem. Objectives: Describe the sociodemographic and clinical characteristics of adolescents with sickle cell disease. Report the prevalence of abnormalities of pubertal development. Identify associated factors that delay pubertal development. Patients and Methods: This was a multicenter analytical cross-sectional study over 7 months at the National Reference Center for Sickle Cell Disease and, at the Brazzaville University Hospital. It concerned adolescents with sickle cell disease aged between 10 to 19 years. The study focused on the sociodemographic characteristics of adolescents, the natural history of sickle cell anemia and the evaluation of secondary sexual characteristics using the Tanner classification. Nutritional status was assessed by calculating body mass index (BMI) and height/age and weight/age ratios. Results: Of the 347 adolescents included, the average age of the adolescents was 15.1 ± 2.5 years, 56.5% had normal puberty, 42.6% had delayed puberty and 0.9% had impuberty. The associated factors were under-nutrition with less than 3 meals/day (p = 0.0000), social status with more marked pubertal delay in orphans (p = 0.00127), more than 5 hospitalizations per year (p = 0.0013), pubertal delay was statistically significant in adolescents who had more than 3 vaso-occlusive crises (p = 0.0000), and those who had more than 5 blood transfusions since the discovery of the disease (p = 0.0127). Conclusion: The factors that hinder pubertal development in sickle cell patients are intrinsic (sickle cell anemia with its complications) and extrinsic (environmental: diet, social status). The appearance of secondary sexual characteristics is delayed on average by two years compared to the general population.

Homozygous Hemoglobinosis CC: A Series of 3 Cases and a Review of the Literature

Hemoglobinosis C occurs mainly in Africa and America with a high frequency in West Africa. In Senegal, homozygous hemoglobinopathy CC constitutes a very rare profile of which only 3 cases are followed in the clinical hematology department of Dakar. The 1st case is a 49-year-old female patient, with notion of 1st degree consanguinity, and a long history of abdominal pain who presented a poorly tolerated anemic syndrome and splenomegaly. The biological assessment showed moderate anemia (7.6 g/dL) with microcytic hypochromia and a CC profile (HbC = 99.2%;HbA2 = 0.8%) on hemoglobin electrophoresis. The second case was a 22-year-old female patient with a notion of 2nd degree consanguinity who presented a Chauffard triad. The haemogram showed mild anaemia (11 g/dL), microcytic and hypochromic. Hemoglobin electrophoresis confirmed a CC profile (HbC = 95.3%;HbA2 = 4.7%). The third patient was 27 years old, with a history of diffuse abdominal pain and 2nd degree consanguinity. The haemogram and haemoglobin electrophoresis confirmed the CC profile (HbC = 94.6%;HbA2 = 5.4%). The negativity of the Emmel test in front of this presentation suggestive of sickle cell disease means that this type of hemoglobinopathy is diagnosed late in our regions. We therefore recommend the systematic performance of hemoglobin electrophoresis in the presence of any chronic hemolytic anemia.

Hearing analysis in heterozygous and homozygous klotho gene deficient mice

Objective: To understand the crucial role of the klotho gene in hearing development in mouse models.Methods: PCR was used to identify CBA mice with different genotypes, i.e. WT, heterozygous(klotho +/-)or homozygous(klotho-/-). Mice phenotype and weight were recorded postnatal 25 days(P-25) and auditory brainstem responses(ABR) were used to determine auditory function at P-60.Results: klotho-/-mice tended to have smaller size, lighter weight and higher ABR thresholds at P-60,showing early onset age-related hearing loss(ARHL).Conclusion: Heterozygous and homozygous klotho deficient mice exhibit different degrees of hearing loss at young age, with homozygous mice(klotho-/-) showing more severe hearing loss. Our results indicate that persisted expression of klotho protein in the inner ear may potentially delay the onset of ARHL and play an important role in the protection of auditory function.

Gitelman syndrome caused by a rare homozygous mutation in the SLC12A3 gene:A case report

BACKGROUND Gitelman syndrome(GS)is an unusual,autosomal recessive salt-losing tubulopathy characterized by hypokalemic metabolic alkalosis,hypomagnesemia and hypocalciuria.It is caused by mutations in the solute carrier family 12 member 3(SLC12A3)gene resulting in disordered function of the thiazidesensitive NaCl co-transporter.To date,many types of mutations in the SLC12A3 gene have been discovered that trigger different clinical manifestations.Therefore,gene sequencing should be considered before determining the course of treatment for GS patients.CASE SUMMARY A 55-year-old man was admitted to our department due to hand numbness and fatigue.Laboratory tests after admission showed hypokalemia,metabolic alkalosis and renal failure,all of which suggested a diagnosis of GS.Genome sequencing of DNA extracted from the patient’s peripheral blood showed a rare homozygous mutation in the SLC12A3 gene(NM_000339.2:chr16:56903671,Exon4,c.536T>A,p.Val179Asp).This study reports a rare homozygous mutation in SLC12A3 gene of a Chinese patient with GS.CONCLUSION Genetic studies may improve the diagnostic accuracy of Gitelman syndrome and improve genetic counseling for individuals and their families with these types of genetic disorders.

Minor modifications in obtainable Arabidopsis floral dip method enhances transformation efficiency and production of homozygous transgenic lines harboring a single copy of transgene

Many researchers have developed various methods for in-planta or floral dip transformation of Arabidopsis thaliana, one of the simple protocol and widely used to produce transgenic Arabidopsis. As the efficiency and ease of getting a transformant is very much time consuming effort and less number of the transformants people get, we have developed a little modified transformation protocol to avoid the disparities. Four types of inoculums (inoculum1, inoculum2, inoculum3 and inoculum4) were used to check the transformation efficiency out of which Inoculum3 showed the highest rate of transformation among the four types. 0.07% Twin-20 also acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis. After vacuum infiltration keeping the Agrobacterium infected plants for 7-8 hrs horizontally in low light at 280C temperature condition, considered best to get an increased number of transformed seeds. Modified protocol produced ~12-14% increase in transformants. Selection pots (kanamycin supplemented soil filled pots) in place of selection plates (Kanamycin supplemented Murashige and Skoog agar plates) proved beneficial as no MS medium and no aseptic condition is required for selection of transformed plants. This increase in transformation efficiency consequently increased the percentage of homozygous and single copied stable transgenic lines.

Selection of Homozygous Cotton Lines Transformed with Two Insect-Resistant Genes

A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera , nine transgenic homozygous cotton lines with insect resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.

Characterization of coronary atherosclerotic plaques in a homozygous familial hypercholesterolemia visualized by optical coherence tomography

Familial hypercholesterolemia(FH)is an autosomal dominant genetic disorder,which resulted in severe elevations in low-density lipoprotein cholesterol(LDL-C)and a markedly increased risk of early-onset coronary disease.[1]t is most frequently caused by loss-of-function mutations in genes affecting the LDL receptor,which clears LDL particles from plasma.

Rare large homozygous CFTR gene deletion in an Iranian patient with cystic fibrosis

Cystic fibrosis, a common autosomal recessive genetic disorder among Caucasians, is caused by defects in the transmembrane conductance regulatory(CFTR) gene. The analysis of CFTR gene mutations is useful to better characterize the disease, and for preconceptional screening, prenatal and preimplantation genetic diagnosis. Here we report the results of a genetic analysis in a 16-year-old boy from southwestern Iran diagnosed as having cystic fibrosis in infancy based on gastrointestinal and pulmonary manifestations, with positive sweat chloride tests. He lacked both normal and mutant forms of the fragment corresponding to the F508 allele in initial genetic studies. Multiplex ligationdependent probe amplification-based testing revealed a homozygous deletion spanning exons 4 to 10 of the CFTR gene. We predict an in-frame deletion removing 373 amino acids based on our sequencing results. Determining CFTR gene mutations in patients and their family members would be helpful to prevent the occurrence of new cases, especially in populations in which consanguinity is common.

Delivery Outcome in Women with Major Sickle Cell Syndrome: A Comparative Study of the Homozygous Forms “SS” versus the Heterozygous “SC”

Objectives: To determine the prevalence of women who delivered in the two major sickle cell syndromes, “SS” and “SC”, and to identify maternal and early neonatal prognosis inherent to each form. Material and Methods: This is a comparative, descriptive and retrospective cross-sectional study of 226 files of women carrying major sickle cell syndrome (66 cases of “SS” form versus 160 cases of “SC” form), collected from May 2008 to May 2013 at the Gynecology and Obstetrics Clinic of the Sylvanus Olympio’s University Hospital of Lomé. Data were processed by Epi Info 6 software. For comparison of variables, the Chi-2 test of Fisher with significance as p < 0.05 has been used, so is the calculation of Odds Ratio with its confidence interval at 95%. Results: Carriers of the two major sickle cell syndromes represent 0.8% of all the deliveries during the study period. Caesarean section, especially prophylactic one, was the dominant mode of delivery. The SS forms have been exposed to have more vaso-occlusive crises (22.7% vs. 13.1%;p = 0.04, OR = 0.31), more blood transfusion (57.6% vs. 29.4%;p = 7 × 10-5, OR = 3.2) and more puerperal infections (p < 0.05). Acute chest syndrome was not related to any of the two forms of sickle cell disease (13.6% vs. 8.1%;p = 0.15). The maternal mortality rate and the perinatal mortality among SS form against SC form were not significant (respectively 15.1% vs. 8.7%;20.9% vs. 17.1%). Conclusion: Maternal and fetal complications were present in both forms of major sickle cell syndrome but the "SS" form gave exposure to greater maternal morbidity. Resuscitative measures in adults and newborns should be reinforced at the delivery time of these “at-risk-pregnancies”.

Detection of Homozygous Deletions and Mutations in the CDKN2A Gene in Hydatidiform Moles

OBJECTIVE To investigate homozygous deletions and mutations in the CDKN2A gene(p16 INK4a and p14 ARF gene)in hydatidiform moles. METHODS A total of 38 hydatidiform mole samples and 30 villi samples were examined for homozygous deletions in the CDKN2A gene by PCR and for mutations by DHPLC. RESULTS i)Among 38 hydatidiform mole samples, homozygous deletions in the p16 INK4a exon 1 were identified in 5 cases(13.2%),while no homozygous deletions were found in the p16I NK4aexon 1 of 30 early-pregnancy samples.The rates of those deletions in hydatidiform compared to early-pregnancy villi samples was statistically significant(P=0.036).ii)No homozygous deletions in the p14 ARF exon 1 or p16 INK4a exon 2 were found in any of the hydatidiform moles or early-preganancy samples.iii) In all hydatidiform moles and early-pregnancy villi samples,no mutations were detected by DHPLC. CONCLUSION We suggest there may be a close correlation between homozygous deletions in the CDKN2A gene and occurrence of hydatidiform moles variation in the CDKN2A gene is mainly caused by homozygous deletions,while mutations may be not a major cause.

The Mesenteric-Caval Fistula: First Results of a New Technique in a Transperitoneal Reconstruction of the Caval Vein by Fulminant Thrombosis of the Inferior Vena Cava Based on Homozygous Antithrombin III-Deficiency

Recurrent thrombotic occlusions are one major problem in patients with thrombosis of the inferior vena cava. Due to this, we report a new surgical strategy for the construction of aorto-caval (mesenteric-caval) fistula in a patient with homozygous Antithrombin III (ATIII)-Deficiency. The patient survived postoperatively and only surgical complications grade I and II (Clavien-Dindo classification) were reported after short-term and one year follow-up. After one year, the CT-angiography did not show any caval thrombosis or stenosis and no restriction or occlusion of the fistula. Thus, the mesenteric-caval fistula could be safely performed and resulted in a satisfactory patency.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

A 42-year-old woman with 4H leukodystrophy caused by a homozygous mutation in POLR3A gene

A 42-year-old woman,born to non-consanguineous parents with no family history of neurological diseases,suffered from ataxia for 3 years.After a normal psychomotor development,the patient first presented dental eruption at 1-year-old and with absence at 5 years of age.She stopped growing when she was 15 years old,and remained heights under 150 cm.Intellectual impairment became evident at 35 years old.Approximately 4 years ago (38 years old),she appeared secondary amenorrhea.Ataxia aggravated in the following 3 years and gradually affected daily life.On examinations,she has short stature [Supplementary Figure 1,http://links.lww.com/CM9/A61],tooth dysplasia and ataxia-related syndrome.There were no nystagmus,papilla atrophy or other visual problems.Her Mini-Mental State Examination (MMSE) score was 19,indicating mild intellectual disability.No contraceptives were used.She gave birth to a boy by making a cesarean section at 23-year-old,and her son was without any neurological symptom so far.Her parents died of other internal medicine diseases.Their gonads developed normally when they were alive.Only one brother who showed normal appearance and cognition was in her family.

Morphological characteristics of homozygous wild rice phytoliths and their significance in the study of rice origins

The analysis of wild rice cell structures,tissues,organs,and other morphological characteristics and the development of identification markers for wild rice are the basis for identifying the origins and evolution of prehistorical rice agriculture.However,contemporary wild rice strains are often subject to gene introgression from domesticated rice cultivated by humans during the evolutionary process,which may affect the accuracy of wild rice identification markers.This means that how to eliminate the effects of gene introgression from domesticated rice and other plants on the identification of origin markers,and the purification of the morphological characteristics of wild rice have become critical in research to identify the origin of rice.In this study,we compared and analysed the phytolith morphologies of three common wild rice species(Oryza rufipogon Griff.)from various habitats and one species of ectopically preserved homozygous common wild rice after six consecutive generations of self-crossing.We found that the morphology of the bulliform phytolith in the homozygous wild rice with reduced domestication gene introgression had three significant differences compared with native wild rice:(1)an overall reduction in size(body length decreased from 41.9μm in VLnativeto 38.6μm in VLhomozygous);(2)an increase in the proportion of the long-stalked phenotype,with the ratio of B/A decreasing from 1.22±0.47 in B/Anativeto 0.92±0.30 in B/Ahomozygous;and(3)a decrease in the number of fish-scale decorations,with the proportion of bulliform phytoliths with≥9 fish-scale decorations reduced from 53.4%in native wild rice to 37.2%in homozygous wild rice.Thus,this study provides a reliable reference for the identification of rice origins using rice phytolith morphology.

Nutritional Quality of the Maize Combinations with Mutant Gene opaque-2 (o2)

[Objective] This study aimed to investigate the action of mutant gene o2 and its effect on nutritional quality of different maize combinations. [Method] A total of 33 normal maize combinations from 18 inbred lines were compared with 33 combinations including gene o2 from the corresponding o2 near-isogenic lines （o2-NILs）, to study the effect of o2 gene introduction on maize grain quality. [Result] The contents of lysine, protein and oil in o2-NILs were greatly more than that of normal maize combinations. Except for lysine, contents of other 14 amino acids changed when o2 gene was introduced. Contents of aspartic acid, threonine, glycine, isoleucine, histidine, arginine and proline were improved; while contents of serine, glutamic acid, alanine, valine, leucine, tyrosine and phenylalanine were decreased. Correlation analysis showed that contents of aspartic acid, arginine and threonine had the highest correlation with lysine content. Protein and oil contents had higher correlation with lysine content （0.48 and 0.38）. Analysis of 33 o2-NILs revealed that the o2 combinations CAL58×Ji477and CA156×196 showed high comprehensive quality and high yield with greater development potential. [Conclusion] This study will provide theoretical and material basis for improving the quality of temperate maize germplasm by introducing o2 gene.

Two distinct pathways of p16 gene inactivation in gallbladder cancer

AIM: To examine the mechanism of inactivation of the p16 gene in gallbladder cancer,and to investigate p16 alterations and their correlation with clinicopathological features. METHODS: Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression,loss of heterozygosity (LOH),homozygous deletion and promoter hypermethylation using immunohistochemistry,microsatellite analysis,quantitative real-time polymerase chain reaction (PCR) and methylation-specific PCR,respectively. In addition,mutations were examined by direct DNA sequencing. RESULTS: Homozygous deletions of the p16 gene exon2,LOH at 9p21-22,p16 promoter hypermethylation,and loss of p16 protein expression were detected in 26.0% (13/50),56.9% (29/51),72.5% (37/51) and 62.7% (32/51),respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05). Homozygous deletion of the p16 gene,a combination LOH and promoter hypermethylation,and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression (P < 0.05). LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% (2/13) and 92.3% (12/13) of the tumors with homozygous deletion of the p16 gene,respectively. P16 alterations were not associated with clinicopathological features. CONCLUSION: Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion,a combination of LOH and promoter hypermethylation,and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.