Distinct Phenotypic Variation Research Articles

SSR-BASED ASSESSMENT OF GENETIC DIVERSITY IN BARLEY COLLECTION FROM THE STATE COMMISSION FOR VARIETY TESTING OF AGRICULTURAL CROPS OF KAZAKHSTAN

Barley (Hordeum vulgare L.) is a globally significant cereal crop with a substantial production footprint. As a versatile commodity, it serves as a primary resource for animal feed, malting, and human consumption. Genetic diversity among 49 barley accessions procured from the State Commission for Variety Testing of Agricultural Crops in Kazakhstan was assessed using a set of 21 SSR markers associated with various agronomic traits. The collection exhibited moderate genetic diversity, as indicated by a mean polymorphic information content (PIC) of 0.548 ± 0.153 and a mean Shannon's information index (I) of 0.980 ± 0.317, aligning with previously reported estimates of barley genetic variation in the World. STRUCTURE analysis identified three distinct population clusters (K = 3) using Bayesian clustering and DeltaK, which was confirmed by PCoA results. However, neighbor-joining tree analysis revealed a more detailed population structure with six distinct clusters, indicating substantial genetic diversity within the barley collection. Distinct phenotypic variations were observed among identified genetic clusters, implying a strong association between genetic diversity and agronomic traits, including plant height, kernel number per spike, and grain yield. Seven barley accessions (GSI_B_60, GSI_B_82, GSI_B_216, GSI_B_85, GSI_B_96, GSI_B_141, and GSI_B_208) forming a cluster NJ_tree_2 demonstrated the highest mean grain yield of 50.99 ± 2.95 g/ m2. This specific cluster and its constituent accessions exhibit promising potential for development into high-yielding cultivars or as a valuable source of beneficial alleles for barley breeding initiatives.

Jammed joints and constricted heart: The science of tribology and missing lubricin. A case report on camptodactyly-arthropathy-coxa vara-pericarditis syndrome

ABSTRACT An autosomal recessively inherited noninflammatory arthropathy known as camptodactyly, arthropathy, coxa vara, and pericarditis (CACP) syndrome was discovered in 1999. It is distinguished by synoviocyte hyperplasia and subcapsular fibrosis of the synovial capsule, which results in a shortage of lubricin production. The resulting lack of joint lubrication induces increased mechanical stress, causing progressive deformities that become evident with weight-bearing and heightened joint activity. Animal models with a lubricin gene knock-out display similar traits, underscoring the impact of mechanical stress on disrupting type II collagen on the articular surface. The gradual development of pericarditis and constriction often results in misdiagnosis as juvenile rheumatoid arthritis with cardiac involvement, but the defining feature remains the noninflammatory nature of the disease. Early recognition is pivotal, as interventions such as pericardiectomy and recombinant human lubricin hold promise for altering the disease’s natural course. In our familial case of CACP, two siblings exhibited distinct phenotypic variations – one with fibrosis-dominant features and pericardial constriction and the other displaying synovial hyperplasia without pericardial involvement.

Insights into the mechanism of color formation of the freshwater prawn (Macrobrachium rosenbergii) revealed by de novo assembly transcriptome analysis

Body color is an important visual indicator of crustacean quality and plays a major role in consumer acceptability, perceived quality, and the market price of crustaceans. The freshwater prawn (Macrobrachium rosenbergii) has two distinct phenotypic variations, characterized by dark blue and light yellow body colors. However, the underlying mechanisms regulating the body color of M. rosenbergii remain unclear. In this study, the composition of shell color parameters and pigment cells of raw and cooked dark blue and light yellow M. rosenbergii was investigated and the mechanisms associated with body color were elucidated by transcriptome analysis. The results showed significant differences in the raw shells of the dark blue and light yellow M. rosenbergii (L: 26.20 ± 0.53 vs. 29.25 ± 0.45; a: −0.88 ± 0.19 vs. 0.35 ± 0.18; b: 1.73 ± 0.20 vs. 3.46 ± 0.37; dE: 70.33 ± 0.53 vs. 67.34 ± 0.45, respectively, p = 0.000) as well as the cooked shells (L: 58.14 ± 0.81 vs. 55.78 ± 0.55; a: 19.30 ± 0.56 vs. 16.42 ± 0.40; b: 23.60 ± 0.66 vs. 20.30 ± 0.40, respectively, p < 0.05). Transcriptome differential gene analysis obtained 39.02 Gb of raw data and 158,026 unigenes. Comprehensive searches of the SwissProt, Nr, KEGG, Pfam, and KOG databases resulted in successful annotations of 23,902 (33 %), 40,436 (25.59 %), 32,015 (20.26 %), 26,139 (16.54 %), and 22,155 (14.02 %) proteins, respectively. By KEGG pathway analysis, numerous differentially expressed genes were related to pigmentation-related pathways (MAPK signaling pathway, Wnt signaling pathway, melanin production, tyrosine metabolism, and cell-cell communication process). Candidate DEGs that may be involved in body color included apolipoprotein D, crustacyanin, cytochrome P450, and tyrosinase, as verified by quantitative real-time PCR. The results of this study provide useful references to further elucidate the molecular mechanisms of color formation of M. rosenbergii and other crustaceans.

Multi-generation study of heavy ion beam-induced mutations and agronomic trait variations to accelerate rice breeding.

Heavy ion beam (HIB) is an effective physical mutagen that has been widely used in plant mutational breeding. Systemic knowledge of the effects caused by different HIB doses at developmental and genomic levels will facilitate efficient breeding for crops. Here we examined the effects of HIB systematically. Kitaake rice seeds were irradiated by ten doses of carbon ion beams (CIB, 25 - 300 Gy), which is the most widely used HIB. We initially examined the growth, development and photosynthetic parameters of the M1 population and found that doses exceeding 125 Gy caused significant physiological damages to rice. Subsequently, we analyzed the genomic variations in 179 M2 individuals from six treatments (25 - 150 Gy) via whole-genome sequencing (WGS). The mutation rate peaks at 100 Gy (2.66×10-7/bp). Importantly, we found that mutations shared among different panicles of the same M1 individual are at low ratios, validating the hypothesis that different panicles may be derived from different progenitor cells. Furthermore, we isolated 129 mutants with distinct phenotypic variations, including changes in agronomic traits, from 11,720 M2 plants, accounting for a 1.1% mutation rate. Among them, about 50% possess stable inheritance in M3. WGS data of 11 stable M4 mutants, including three lines with higher yields, reveal their genomic mutational profiles and candidate genes. Our results demonstrate that HIB is an effective tool that facilitates breeding, that the optimal dose range for rice is 67 - 90% median lethal dose (LD50), and that the mutants isolated here can be further used for functional genomic research, genetic analysis, and breeding.

Three-dimensional phenotype characteristics of skeletal class III malocclusion in adult Chinese: a principal component analysis-based cluster analysis.

Skeletal class III malocclusion has a diverse and complicated aetiology involving environmental and genetic factors. It is critical to correctly classify and define this malocclusion to be diagnosed and treated on a clinically sound basis. Thus, this study aimed to provide reliable and detailed measurements in a large ethnically homogeneous sample of Chinese adults to generate an adequate phenotypic clustering model to identify and describe the skeletal variation present in skeletal class III malocclusion. This is a retrospective cross-sectional study in which 500 pre-treatments cone-beam computed tomography (CBCT) scans of patients with skeletal class III malocclusion (250 males and 250 females) were selected following specific selection criteria. Seventy-six linear, angular, and ratios measurements were three-dimensionally analysed using InVivo 6.0.3 software. These measurements were categorised into 47 skeletal, 18 dentoalveolar, and 11 soft tissue variables. Multivariate reduction methods: principal component analyses and cluster analyses were used to present the most common phenotypic groupings of skeletal class III malocclusion in Han ethnic group of Chinese adults. The principal component analysis revealed eight principal components accounted for 72.9% of the overall variation of the data produced from the seventy-six variables. The first four principal components accounted for 53.37% of the total variations. They explained the most variation in data and consisted mainly of anteroposterior and vertical skeletal relationships. The cluster analysis identified four phenotypes of skeletal class III malocclusion: C1, 34%; C2, 11.4%; C3, 26.4%; and C4, 28.2%. Based on three-dimensional analyses, four skeletal class III malocclusion distinct phenotypic variations were defined in a large sample of the adult Chinese population, showing the occurrence of phenotypic variation between identified clusters in the same ethnic group. These findings might serve as a foundation for accurate diagnosis and treatment planning of each cluster and future genetic studies to determine the causative gene(s) of each cluster.

Four Looks to a Gene (P1-8.013)

<h3>Objective:</h3> We present a family with varied clinical presentations of a genetically confirmed muscular dystrophy. <h3>Background:</h3> A 41-year-old male presented with several years of muscle fatigue, pain and twitching across his chest and proximal muscles. He was diagnosed with neuronal hyperexcitability. Exam showed normal muscle strength, but there were visible rippling in proximal muscles. Cardiac workup was unrevealing. EMG showed nonspecific myopathic features and CPK was in the 400’s. His 9-year-old son has been diagnosed with muscular dystrophy and two daughters has elevated CPKs. Our patient was genetically confirmed to have a heterozygous C.80G&gt;A(p.Arg27Gln) mutation in the Caveolin-3 gene via Invitae muscular dystrophy testing. This missense mutation causes an amino acid change from Arg to Gln in the Caveolin 3 protein, which leads to a decrease of Cav-3 protein expression in skeletal muscle tissue. It has an autosomal dominant inheritance pattern. <h3>Design/Methods:</h3> Not applicable <h3>Results:</h3> Mutations in the Caveolin-3 gene have been described in a broad spectrum of clinical presentations including limb girdle muscular dystrophy, rippling muscle disease, idiopathic persistent hyperCKemia, and distal myopathy. Muscular dystrophy can present with a wide variety of phenotypes and there is even variation within the same gene mutations, as is the case in caveolin 3 mutations. This may lead to delay in diagnosis and supportive treatments that a family may require. The Invitae muscular dystrophy panel can be helpful in expediting diagnosis for multiple family members. It is also important to remember that muscular dystrophy can mimic muscle hyperexcitability syndrome, as was the case in our patient with rippling muscle disease. <h3>Conclusions:</h3> Muscular dystrophy can present with distinct phenotypic variations. A good family history and awareness of clinical symptoms can be helpful for diagnosis. CAV3 mutations cause LGMD1C which can present with at least four different phenotypes. <b>Disclosure:</b> Dr. Rahimi has nothing to disclose. Dr. Memar Ardestani has nothing to disclose. Dr. Prabhu has nothing to disclose. Dr. Undavia has nothing to disclose.

Distinct Phenotypic Variation of Blastocystis sp. ST3 from Urban and Orang Asli Population-An Influential Consideration during Sample Collection in Surveys.

Simple SummaryBlastocystis sp. is a common intestinal protozoan of humans with the phenotypic characteristics strongly associated with its activity, including pathogenicity. This characteristic varies, but the variation has not been clearly understood. The present study evaluates the variation when a single subtype of Blastocystis sp. was isolated from a population with distinct gut microbial composition, namely, the urban and orang asli(indigenous) population. Blastocystis sp. cells isolated from orang asli individuals had a higher growth rate with elevated resistance to harsh conditions. Distinct surface coats with amoebic forms were noticed in parasite cells from urban individuals. Proteases, commonly a virulent factor in other parasites, showed variation depending on the isolation source. Stimulation of cancer cell proliferation by only Blastocystis sp. isolated from urban individuals is suggestive of the variation at the antigenic level. This phenotypic variation suggests that implicating subtype to pathogenicity may be too early, and a deeper understanding of Blastocystis sp. and microenvironment interaction is essential.Blastocystis sp. is a globally distributed protozoan parasite with uncertain pathogenicity. Phenotypic variation in Blastocystis sp. suggests its adaptation; however, the phenotypic features of Blastocystis sp. ST3 from a distinct source of isolation is unknown. Blastocystis sp. isolated from individuals in urban and orang asli (indigenous population in Selangor, Malaysia) settlements were studied for phenotypic characteristics such as growth profile, morphology, ultrastructure, and resistance to harsh conditions. Subsequently, pathogenic potentials, such as in protease activity and the ability to stimulate the proliferation of cancer cells, were assessed. Higher parasite counts with granular and apoptotic forms were found in Blastocystis sp. from orang asli individuals. Cells with fuzzy coats and amoebic structures which seemingly implicate increased interaction with bacteria were seen predominantly in urban symptomatic persons. Also, Blastocystis sp. from orang asli isolates resisted harsh environments, suggesting longer co-adaptation to the hosts. Urban and orang asli symptomatic isolates possessed a predominance of only cysteine protease, whereas all the asymptomatic isolates showed significantly higher cysteine, serine, or aspartic protease activity. However, only solubilized antigen from urban symptomatic isolates showed significant stimulation of cancer cell proliferation. For the first time, our findings demonstrate significant phenotypic variation in a single subtype, ST3 of Blastocystis sp., isolated from urban and orang asli populations that are known to have distinct gut microbial compositions. The outcome emphasizes the importance of identifying people’s locations and lifestyles during sample collection before forming conclusions on the prevailing data and implicating subtypes to pathogenicity. The environment plays a significant role in Blastocystis sp. infection.

Ancient introgression underlying the unusual mito‐nuclear discordance and coat phenotypic variation in the Moupin pika

AbstractAimThe mechanisms by which global biodiversity hotspots harbour and conserve high genetic and morphological diversity of endemic species remain unexplored. Relic species of ochotonids in the genus Ochotona are confined to alpine habitats and highly sensitive to environmental changes. We studied the genomic and ecological mechanisms underlying the divergence and adaptive evolution of the Moupin pika (O. thibetana) and its closely related species to infer its diversification, adaptive evolution and demographic history in response to historical and recent environmental changes.LocationHengduan Mountain Region, China.MethodsWe integrated morphological, genomic and ecological data to interpret the phylogeographic structure, adaptive evolution, demographic history and range shift of O. thibetana. Phylogenetic reconstruction was based on Cytochrome b (CYTB), the mitochondrial genome and single‐copy orthologues of the whole genome. Gene flow among extant lineages, as well as from extinct species, was inferred by multiple algorithms. Demographic history was inferred using the pairwise sequential Markovian coalescent and high‐resolution analysis of linkage disequilibrium. We also predicted the range shift of this species by using ecological niche modelling.ResultsPhylogenetic reconstruction revealed an unusual mitochondrial lineage of O. thibetana from the western Sichuan Basin, which was named O. qionglaiensis in previous studies. Extensive gene flow was detected among genetic lineages of O. thibetana, which has distinct phenotypic variation in hair thickness and colouration, as well as notable morphological differentiation in external and craniodental measurements. Multiple members of the keratin gene family were identified as introgressed loci from some ancient species to O. thibetana. The Moupin pika underwent dramatic population shrinkage in the late Quaternary, with a clear trend of population growth in approximately the last 70 generations. The potential distribution range of O. thibetana showed a clear trend of expansion in the future.Main conclusionsThe unusual mitochondrial phylogenetic pattern of O. thibetana resulted from “extended ghost introgression,” a new evolutionary model proposed in the present study, and thus rejected the validity of O. qionglaiensis as an independent species. Ancient introgression of keratin genes likely underlies the prominent coat phenotypic variation among genetic lineages. Clear population growth and range expansion of the Moupin pika in recent generations probably benefitted from recent global warming and vegetation recovery, which not only contributed to the conservation of large mammals but was also beneficial to small mammals endemic to alpine habitats.

Two ploidy levels present in the invasive Campuloclinium macrocephalum (pompom weed) in South Africa – Implications for biocontrol

AbstractThe highly invasive Campuloclinium macrocephalum (Asteraceae; ‘pompom weed’), native to South America, was introduced to South Africa where it has flourished and now threatens the native diversity of the grasslands it has invaded. The Argentine C. macrocephalum population is known to comprise at least three ploidy levels, and exhibit aneuploidy. Ploidy levels had not yet been assessed across South African populations of pompom weed and could influence the effectiveness of a biological control (biocontrol) programme. Therefore, we used flow cytometry to estimate ploidy levels of individuals from invaded regions in South Africa and Argentina. Diploids, triploids, tetraploids and aneuploids were detected in the sampled Argentine individuals. In South Africa, only triploid and tetraploid cytotypes were identified, with possible aneuploid variation. Mapping the distribution of these ploidy levels in South Africa revealed that the tetraploids are more widespread across all five sampled provinces, whereas the triploids occur only in Gauteng (the likely origin of the invasion) and neighbouring North West provinces and one sampled area in KwaZulu‐Natal province. Distinct phenotypic variation is evident between Argentine individuals of different ploidy levels. Polyploids have now been confirmed to occur in both the native and introduced ranges. Because triploids and tetraploids are present in both native and introduced ranges, we predict that polyploidy is not likely to be a constraint in effective biocontrol in South Africa as the natural enemies have probably co‐evolved with polyploids in the native range. However, other factors may impact the effectiveness of the biocontrol agents, such as feeding, oviposition (potentially affecting their reproduction), establishment, and spread, therefore, warranting further research.

Metamorphosis of a butterfly inversion.

Chromosomal inversions frequently underlie distinct phenotypic variation. A new study shows that in butterflies, inversion haplotypes accumulate deleterious mutations that prevent fixation in natural populations.

The eukaryotic MEP-pathway genes are evolutionarily conserved and originated from Chlaymidia and cyanobacteria

BackgroundIsoprenoids are the most ancient and essential class of metabolites produced in all organisms, either via mevalonate (MVA)-and/or methylerythritol phosphate (MEP)-pathways. The MEP-pathway is present in all plastid-bearing organisms and most eubacteria. However, no comprehensive study reveals the origination and evolutionary characteristics of MEP-pathway genes in eukaryotes.ResultsHere, detailed bioinformatics analyses of the MEP-pathway provide an in-depth understanding the evolutionary history of this indispensable biochemical route, and offer a basis for the co-existence of the cytosolic MVA- and plastidial MEP-pathway in plants given the established exchange of the end products between the two isoprenoid-biosynthesis pathways. Here, phylogenetic analyses establish the contributions of both cyanobacteria and Chlamydiae sequences to the plant’s MEP-pathway genes. Moreover, Phylogenetic and inter-species syntenic block analyses demonstrate that six of the seven MEP-pathway genes have predominantly remained as single-copy in land plants in spite of multiple whole-genome duplication events (WGDs). Substitution rate and domain studies display the evolutionary conservation of these genes, reinforced by their high expression levels. Distinct phenotypic variation among plants with reduced expression levels of individual MEP-pathway genes confirm the indispensable function of each nuclear-encoded plastid-targeted MEP-pathway enzyme in plant growth and development.ConclusionCollectively, these findings reveal the polyphyletic origin and restrict conservation of MEP-pathway genes, and reinforce the potential function of the individual enzymes beyond production of the isoprenoids intermediates.

Development of Root Phenotyping Platforms for Identification of Root Architecture Mutations in EMS-Induced and Low-Path-Sequenced Sorghum Mutant Population

Sorghum’s natural adaptation to a wide range of abiotic stresses provides diverse genetic reserves for potential improvement in crop stress tolerance. Growing interest in sorghum research has led to the expansion of genetic resources though establishment of the sorghum association panel (SAP), generation of mutagenized populations, and recombinant inbred line (RIL) populations, etc. Despite rapid improvement in biotechnological tools, lack of efficient phenotyping platforms remains one of the major obstacles in utilizing these genetic resources. Scarcity of efforts in root system phenotyping hinders identification and integration of the superior root traits advantageous to stress tolerance. Here, we explored multiple approaches in root phenotyping of an ethyl methanesulfonate (EMS)-mutagenized sorghum population. Paper-based growth pouches (PGP) and hydroponics were employed to analyze root system architecture (RSA) variations induced by mutations and to test root development flexibility in response to phosphorus deficiency in early growing stages. PGP method had improved capabilities compared to hydroponics providing inexpensive, space-saving, and high-throughput phenotyping of sorghum roots. Preliminary observation revealed distinct phenotypic variations which were qualitatively and quantitatively systemized for association analysis. Phenotypes/ideotypes with root architecture variations potentially correlated with Pi acquisition were selected to evaluate their contribution to P-efficiency (PE). Sand mixed with P-loaded activated alumina substrate (SAS) provided closely to natural but still controlled single-variable conditions with regulated Pi availability. Due to higher labor and cost input we propose SAS to be used for evaluating selected sorghum candidates for PE. The ability of rapidly screening root phenotypes holds great potential for discovering genes responsible for relevant root traits and utilizing mutations to improve nutrient efficiency and crop productivity.

Distinct Splice Variants and Pathway Enrichment in the Cell-Line Models of Aggressive Human Breast Cancer Subtypes

This study was conducted as a part of the Chromosome-Centric Human Proteome Project (C-HPP) of the Human Proteome Organization. The United States team of C-HPP is focused on characterizing the protein-coding genes in chromosome 17. Despite its small size, chromosome 17 is rich in protein-coding genes; it contains many cancer-associated genes, including BRCA1, ERBB2, (Her2/neu), and TP53. The goal of this study was to examine the splice variants expressed in three ERBB2 expressed breast cancer cell-line models of hormone-receptor-negative breast cancers by integrating RNA-Seq and proteomic mass spectrometry data. The cell lines represent distinct phenotypic variations subtype: SKBR3 (ERBB2+ (overexpression)/ER-/PR-; adenocarcinoma), SUM190 (ERBB2+ (overexpression)/ER-/PR-; inflammatory breast cancer), and SUM149 (ERBB2 (low expression) ER-/PR-; inflammatory breast cancer). We identified more than one splice variant for 1167 genes expressed in at least one of the three cancer cell lines. We found multiple variants of genes that are in the signaling pathways downstream of ERBB2 along with variants specific to one cancer cell line compared with the other two cancer cell lines and with normal mammary cells. The overall transcript profiles based on read counts indicated more similarities between SKBR3 and SUM190. The top-ranking Gene Ontology and BioCarta pathways for the cell-line specific variants pointed to distinct key mechanisms including: amino sugar metabolism, caspase activity, and endocytosis in SKBR3; different aspects of metabolism, especially of lipids in SUM190; cell-to-cell adhesion, integrin, and ERK1/ERK2 signaling; and translational control in SUM149. The analyses indicated an enrichment in the electron transport chain processes in the ERBB2 overexpressed cell line models and an association of nucleotide binding, RNA splicing, and translation processes with the IBC models, SUM190 and SUM149. Detailed experimental studies on the distinct variants identified from each of these three breast cancer cell line models that may open opportunities for drug target discovery and help unveil their specific roles in cancer progression and metastasis.

Major-Effect Alleles at Relatively Few Loci Underlie Distinct Vernalization and Flowering Variation in Arabidopsis Accessions

We have explored the genetic basis of variation in vernalization requirement and response in Arabidopsis accessions, selected on the basis of their phenotypic distinctiveness. Phenotyping of F2 populations in different environments, plus fine mapping, indicated possible causative genes. Our data support the identification of FRI and FLC as candidates for the major-effect QTL underlying variation in vernalization response, and identify a weak FLC allele, caused by a Mutator-like transposon, contributing to flowering time variation in two N. American accessions. They also reveal a number of additional QTL that contribute to flowering time variation after saturating vernalization. One of these was the result of expression variation at the FT locus. Overall, our data suggest that distinct phenotypic variation in the vernalization and flowering response of Arabidopsis accessions is accounted for by variation that has arisen independently at relatively few major-effect loci.

Variability in cognitive presentation of Alzheimer's disease

The aim of the present study was to explore the nature and prevalence of phenotypic variations in Alzheimer's disease (AD). Neuropsychological profiles of a large cross-sectional cohort of patients with a clinical diagnosis of the disease were examined. All tests distinguished the AD group from controls confirming their sensitivity to the presence of early AD. Factor analysis of test scores revealed five factors, reflecting the discrete cognitive domains of memory, language, perceptuospatial abilities, executive skills, and praxis. Cluster analysis revealed distinct performance profiles that could not be accounted for by disease severity. Some patients showed an accentuation of memory impairment relative to other domains, whereas others showed relative sparing. Cognitive deficits other than memory were the salient presenting feature in a relatively high proportion of patients. A subset of the cohort (22%) showed grossly disproportionate impairments in one cognitive domain. The findings emphasise variability in presentation and indicate that distinct phenotypic variations appear to lie on a continuum rather than representing discrete forms of disease.

Phenotypic Variation in a Family with Partial Androgen Insensitivity Syndrome Explained by Differences in 5α Dihydrotestosterone Availability

Mutations in the androgen receptor (AR) gene result in a wide range of phenotypes of the androgen insensitivity syndrome (AIS). Inter- and intrafamilial differences in the phenotypic expression of identical AR mutations are known, suggesting modifying factors in establishing the phenotype. Two 46,XY siblings with partial AIS sharing the same AR gene mutation, R846H, but showing very different phenotypes are studied. Their parents are first cousins. One sibling with grade 5 AIS was raised as a girl; the other sibling with grade 3 AIS was raised as a boy. In both siblings serum levels of hormones were measured; a sex hormone-binding globulin (SHBG) suppression test was completed; and mutation analysis of the AR gene, Scatchard, and SDS-PAGE analysis of the AR protein was performed. Furthermore, 5alpha-reductase 2 expression and activity in genital skin fibroblasts were investigated, and the 5alpha-reductase 2 gene was sequenced. The decrease in SHBG serum levels in a SHBG suppression test did not suggest differences in androgen sensitivity as the cause of the phenotypic variation. Also, androgen binding characteristics of the AR, AR expression levels, and the phosphorylation pattern of the AR on hormone binding were identical in both siblings. However, 5alpha-reductase 2 activity was normal in genital skin fibroblasts from the phenotypic male patient but undetectable in genital skin fibroblasts from the phenotypic female patient. The lack of 5alpha-reductase 2 activity was due to absent or reduced expression of 5alpha-reductase 2 in genital skin fibroblasts from the phenotypic female patient. Exon and flanking intron sequences of the 5alpha-reductase 2 gene showed no mutations in either sibling. Additional intragenic polymorphic marker analysis gave no evidence for different inherited alleles for the 5alpha-reductase 2 gene in the two siblings. Therefore, the absent or reduced expression of 5alpha-reductase 2 is likely to be additional to the AIS. Distinct phenotypic variation in this family was caused by 5alpha-reductase 2 deficiency, additional to AIS. This 5alpha-reductase deficiency is due to absence of expression of the 5alpha-reductase iso-enzyme 2 as shown by molecular studies. The distinct phenotypic variation in AIS here is explained by differences in the availability of 5alpha-dihydrotestosterone during embryonic sex differentiation.